Opioid receptor antagonist prodrugs

ABSTRACT

Provided herein are prodrugs of opioid receptor antagonists such as nalmefene and naltrexone, pharmaceutical compositions comprising said compounds, and methods for using said compounds for the treatment of behavioral disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of International Application No. PCT/CN2018/113850, filed on Nov. 3, 2018, which claims benefit of U.S. Provisional Patent Application No. 62/581,504 filed on Nov. 3, 2017, and U.S. Provisional Patent Application No. 62/697,289 filed on Jul. 12, 2018. The entire contents of the aforementioned application are incorporated herein by reference.

BACKGROUND

A need exists in the medicinal arts for compositions and methods for the modulation of opioid receptor activity in the course of treating behavioral disorders.

BRIEF SUMMARY OF THE INVENTION

Provided herein are prodrugs of opioid receptor antagonists such as nalmefene and naltrexone, pharmaceutical compositions comprising said compounds, and methods for using said compounds for the treatment of behavioral disorders.

Some compounds of the invention have superior properties. For example, some compounds of the invention have superior stabilities in oil based pharmaceutical compositions such as sesame oil or cottonseed oil.

Some compounds of the invention have better pharmacokinetic activities in vivo (for example, rat or dog), e.g., extended half-life.

Some compounds of the invention have better safety in vivo (for example, rat or dog), e.g., diminished injection site reactions.

Some compounds of the invention have superior stability either neat or in oil based pharmaceutical compositions.

In one aspect, provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (I),

wherein,

X is O or CH₂;

R is selected from:

a. (C₃-C₇cycloalkyl)CH₂C(O)—;

b. (C₃-C₇cycloalkyl)CH₂CH₂C(O)—;

c. —C(O)OC₇-C₂₀ alkyl; or

d. —C(O)NHC(CH₃)₃.

In some embodiments, X is O. In some embodiments, X is CH₂.

In another aspect, also provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (II),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 7-15; provided if X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In one aspect, provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (II),

wherein,

X is O or CH₂;

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 7-15; provided if X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In another aspect, also provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (IIa),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 9-15.

In some embodiments, X is O. In some embodiments, X is CH₂.

In another aspect, also provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (III),

wherein,

X is O or CH₂;

R is selected from:

-   -   —[CH(R³)O]z-R⁴;     -   —[CH(R³)O]z-C(═O)OR⁴;     -   —[CH(R³)O]z-C(═O)NR⁴R⁵; and     -   —[CH(R³)O]z-P(═O)(OR⁴)(OR⁵);

wherein z is 1, 2, 3, 4, 5, 6, or 7;

R³ is hydrogen, halogen, alkyl, alkenyl, cycloalkylalkyl, or aryl;

-   -   each R⁴ and R⁵ is independently selected from hydrogen, alkyl,         alkenyl, cycloalkylalkyl, or aryl.

In some embodiments, X is O. In some embodiments, X is CH₂.

One embodiment provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof.

Provided herein is a method of treating opioid dependence in a patient in need thereof comprising administering a pharmaceutical composition comprising a compound of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

One embodiment provides a method of treating a patient wherein the therapeutic effect of a long acting opioid antagonist depot can be overcome in a patient by administering an opioid based analgesic.

One embodiment provides a method of treating opioid dependence in a patient in need thereof, wherein the patient receives a first injection of an injectable formulation comprising a compound of any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, wherein said first injection provides a therapeutically relevant plasma concentration for about 1 week, about 2 weeks, about 3 weeks or about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months or at least about 6 months, followed by a second injection of an injectable formulation comprising a compound of any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, wherein said second injection provides a therapeutically relevant plasma concentration for at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months or at least about 6 months.

One embodiment provides a method of treating opioid dependence in a patient in need thereof, wherein the patient receives a first injection of an injectable formulation of naltrexone loaded PLGA microspheres that provides a therapeutically relevant plasma concentration for about 4 weeks, followed by one or more injections of an injectable formulation comprising a compound of any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, that provides a therapeutically relevant plasma concentration for about 2 months, about 3 months, about 4 months, or about 5 months or more.

One embodiment provides a method of treating opioid dependence in a patient in need thereof, wherein the patient receives one or more injections of an injectable formulation comprising at least one compound of any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, wherein the patient has been previously treated with opioid agonists or partial agonists, such as buprenorphine or methadone, and the patients are now transitioning to discontinuation from such agonist or partial agonist treatment.

One embodiment provides a method of treating opioid dependence in a patient in need thereof, wherein the patient receives one or more injections of an injectable formulation comprising at least one compound of any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof, wherein the patient is recently addicted and naïve to prior medication assisted treatment, or wherein the patient has recently discontinued opioid pain medication, are at risk of future opioid drug abuse, and are in need of prevention of future opioid drug abuse via antagonist treatment.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference for the specific purposes identified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings below. The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 provides the nuclear magnetic resonance spectrum of Example 1 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl ((E)-octadec-9-en-1-yl) carbonate;

FIG. 2 provides the nuclear magnetic resonance spectrum of Example 2 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl undecyl carbonate;

FIG. 3 provides the nuclear magnetic resonance spectrum of Example 3 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl undecyl carbonate;

FIG. 4 provides the nuclear magnetic resonance spectrum of Example 4 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl undecyl carbonate; and

FIG. 5 provides the nuclear magnetic resonance spectrum of Example 5 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl undecyl carbonate.

FIG. 6 provides the nuclear magnetic resonance spectrum of Example 6 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate;

FIG. 7 provides the nuclear magnetic resonance spectrum of Example 7 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl ((E)-octadec-9-en-1-yl) carbonate;

FIG. 8 provides the nuclear magnetic resonance spectrum of Example 8 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (E)-octadec-9-enoate;

FIG. 9 provides the nuclear magnetic resonance spectrum of Example 9 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl (E)-octadec-9-enoate;

FIG. 10 provides the nuclear magnetic resonance spectrum of Example 10 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate;

FIG. 11 provides the nuclear magnetic resonance spectrum of Example 11 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate;

FIG. 12 provides the nuclear magnetic resonance spectrum of Example 12 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl stearate;

FIG. 13 provides the nuclear magnetic resonance spectrum of Example 13 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (Z)-docos-13-enoate;

FIG. 14 provides the nuclear magnetic resonance spectrum of Example 14 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl docosanoate;

FIG. 15 provides the nuclear magnetic resonance spectrum of Example 15 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (E)-octadec-9-enoate;

FIG. 16 provides the nuclear magnetic resonance spectrum of Example 16 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl icosanoate;

FIG. 17 provides the nuclear magnetic resonance spectrum of Example 17 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl octyl carbonate;

FIG. 18 provides the nuclear magnetic resonance spectrum of Example 18 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate;

FIG. 19 provides the nuclear magnetic resonance spectrum of Example 19 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate;

FIG. 20 provides the nuclear magnetic resonance spectrum of Example 20 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate;

FIG. 21 provides the nuclear magnetic resonance spectrum of Example 21 of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate;

FIG. 22 provides the nuclear magnetic resonance spectrum of Example 22 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (Z)-docos-13-enoate;

FIG. 23 provides the nuclear magnetic resonance spectrum of Example 23 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl octyl carbonate;

FIG. 24 provides the nuclear magnetic resonance spectrum of Example 24 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl dodecyl carbonate;

FIG. 25 provides the nuclear magnetic resonance spectrum of Example 25 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl tetradecyl carbonate;

FIG. 26 provides the nuclear magnetic resonance spectrum of Example 26 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl (E)-octadec-9-enoate;

FIG. 27 provides the nuclear magnetic resonance spectrum of Example 27 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl tetradecyl carbonate;

FIG. 28 provides the nuclear magnetic resonance spectrum of Example 28 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl icosyl carbonate;

FIG. 29 provides the nuclear magnetic resonance spectrum of Example 29 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl dodecyl carbonate;

FIG. 30 provides the nuclear magnetic resonance spectrum of Example 30 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl tridecyl carbonate;

FIG. 31 provides the nuclear magnetic resonance spectrum of Example 31 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl tetradecyl carbonate;

FIG. 32 provides the nuclear magnetic resonance spectrum of Example 32 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl pentadecyl carbonate;

FIG. 33 provides the nuclear magnetic resonance spectrum of Example 33 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl octadecyl carbonate;

FIG. 34 provides the nuclear magnetic resonance spectrum of Example 34 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl hexadecyl carbonate;

FIG. 35 provides the nuclear magnetic resonance spectrum of Example 35 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl decyl carbonate;

FIG. 36 provides the nuclear magnetic resonance spectrum of Example 36 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl oleate;

FIG. 37 provides the nuclear magnetic resonance spectrum of Example 37 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z)-octadeca-9,12-dienoate;

FIG. 38 provides the nuclear magnetic resonance spectrum of Example 38 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3,3-dimethylbutanoate;

FIG. 39 provides the nuclear magnetic resonance spectrum of Example 39 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3-cyclopentylpropanoate;

FIG. 40 provides the nuclear magnetic resonance spectrum of Example 40 (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl tert-butylcarbamate;

FIG. 41 provides the nuclear magnetic resonance spectrum of Example 41 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl oleate;

FIG. 42 provides the nuclear magnetic resonance spectrum of Example 42 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3,3-dimethylbutanoate;

FIG. 43 provides the nuclear magnetic resonance spectrum of Example 43 (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3-cyclopentylpropanoate;

FIG. 44 provides the nuclear magnetic resonance spectrum of Example 44 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl dodecanoate;

FIG. 45 provides the nuclear magnetic resonance spectrum of Example 45 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl tetradecanoate;

FIG. 46 provides the nuclear magnetic resonance spectrum of Example 46 (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl hexadecanoate.

FIG. 47 provides the nuclear magnetic resonance spectrum of Example 47 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl hexadecyl carbonate;

FIG. 48 provides the nuclear magnetic resonance spectrum of Example 48 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl dodecanoate; and

FIG. 49 provides the nuclear magnetic resonance spectrum of Example 49 (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl hexadecanoate.

DETAILED DESCRIPTION OF THE INVENTION

As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents, and reference to “the cell” includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, “consist of” or “consist essentially of” the described features.

Definitions

As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.

“Amino” refers to the —NH₂ radical.

“Cyano” refers to the —CN radical.

“Nitro” refers to the —NO₂ radical.

“Oxa” refers to the —O— radical.

“Oxo” refers to the ═O radical.

“Thioxo” refers to the ═S radical.

“Imino” refers to the ═N—H radical.

“Oximo” refers to the ═N—OH radical.

“Hydrazino” refers to the ═N—NH₂ radical.

“Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to fifteen carbon atoms (e.g., C₁-C₁₅ alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C₁-C₁₃ alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C₁-C₈ alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (e.g., C₁-C₅ alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (e.g., C₁-C₄ alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (e.g., C₁-C₃ alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (e.g., C₁-C₂ alkyl).

In other embodiments, an alkyl comprises one carbon atom (e.g., C₁ alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C₅-C₁₅ alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C₅-C₈ alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (e.g., C₂-C₅ alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (e.g., C₃-C₅ alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

“Alkoxy” refers to a radical bonded through an oxygen atom of the formula —O-alkyl, where alkyl is an alkyl chain as defined above.

“Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from two to twelve carbon atoms. In certain embodiments, an alkenyl comprises two to eight carbon atoms. In other embodiments, an alkenyl comprises two to four carbon atoms. The alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

“Alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, having from two to twelve carbon atoms. In certain embodiments, an alkynyl comprises two to eight carbon atoms. In other embodiments, an alkynyl comprises two to six carbon atoms. In other embodiments, an alkynyl comprises two to four carbon atoms. The alkynyl is attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

“Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation and having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond.

The points of attachment of the alkylene chain to the rest of the molecule and to the radical group is through one carbon in the alkylene chain or through any two carbons within the chain. In certain embodiments, an alkylene comprises one to eight carbon atoms (e.g., C₁-C₈ alkylene). In other embodiments, an alkylene comprises one to five carbon atoms (e.g., C₁-C₅ alkylene). In other embodiments, an alkylene comprises one to four carbon atoms (e.g., C₁-C₄ alkylene). In other embodiments, an alkylene comprises one to three carbon atoms (e.g., C₁-C₃ alkylene). In other embodiments, an alkylene comprises one to two carbon atoms (e.g., C₁-C₂ alkylene). In other embodiments, an alkylene comprises one carbon atom (e.g., C₁ alkylene). In other embodiments, an alkylene comprises five to eight carbon atoms (e.g., C₅-C₈ alkylene). In other embodiments, an alkylene comprises two to five carbon atoms (e.g., C₂-C₅ alkylene). In other embodiments, an alkylene comprises three to five carbon atoms (e.g., C₃-C₅ alkylene). Unless stated otherwise specifically in the specification, an alkylene chain is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

“Alkynylene” or “alkynylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon triple bond, and having from two to twelve carbon atoms. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. In certain embodiments, an alkynylene comprises two to eight carbon atoms (e.g., C₂-C₈ alkynylene). In other embodiments, an alkynylene comprises two to five carbon atoms (e.g., C₂-C₅ alkynylene). In other embodiments, an alkynylene comprises two to four carbon atoms (e.g., C₂-C₄ alkynylene). In other embodiments, an alkynylene comprises two to three carbon atoms (e.g., C₂-C₃ alkynylene). In other embodiments, an alkynylene comprises two carbon atom (e.g., C₂ alkylene). In other embodiments, an alkynylene comprises five to eight carbon atoms (e.g., C₅-C₅ alkynylene). In other embodiments, an alkynylene comprises three to five carbon atoms (e.g., C₃-C₅ alkynylene). Unless stated otherwise specifically in the specification, an alkynylene chain is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

“Aryl” refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) it-electron system in accordance with the Hickel theory. The ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene.

Unless stated otherwise specifically in the specification, the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant to include aryl radicals optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each R^(b) is independently a direct bond or a straight or branched alkylene or alkenylene chain, and R^(c) is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

“Aralkyl” refers to a radical of the formula —R^(c)-aryl where R^(c) is an alkylene chain as defined above, for example, methylene, ethylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain. The aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.

“Aralkenyl” refers to a radical of the formula —R^(d)-aryl where R^(d) is an alkenylene chain as defined above. The aryl part of the aralkenyl radical is optionally substituted as described above for an aryl group. The alkenylene chain part of the aralkenyl radical is optionally substituted as defined above for an alkenylene group.

“Aralkynyl” refers to a radical of the formula —R^(e)-aryl, where R^(e) is an alkynylene chain as defined above. The aryl part of the aralkynyl radical is optionally substituted as described above for an aryl group. The alkynylene chain part of the aralkynyl radical is optionally substituted as defined above for an alkynylene chain.

“Aralkoxy” refers to a radical bonded through an oxygen atom of the formula —O—R^(c)-aryl where R^(c) is an alkylene chain as defined above, for example, methylene, ethylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain. The aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.

“Carbocyclyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which includes fused or bridged ring systems, having from three to fifteen carbon atoms. In certain embodiments, a carbocyclyl comprises three to ten carbon atoms. In other embodiments, a carbocyclyl comprises five to seven carbon atoms. The carbocyclyl is attached to the rest of the molecule by a single bond. Carbocyclyl is saturated (i.e., containing single C—C bonds only) or unsaturated (i.e., containing one or more double bonds or triple bonds). A fully saturated carbocyclyl radical is also referred to as “cycloalkyl.” Examples of monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated carbocyclyl is also referred to as “cycloalkenyl.” Examples of monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, the term “carbocyclyl” is meant to include carbocyclyl radicals that are optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each R^(b) is independently a direct bond or a straight or branched alkylene or alkenylene chain, and R^(c) is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

“Carbocyclylalkyl” refers to a radical of the formula —R^(c)-carbocyclyl where R^(c) is an alkylene chain as defined above. The alkylene chain and the carbocyclyl radical is optionally substituted as defined above.

“Carbocyclylalkynyl” refers to a radical of the formula —R^(c)-carbocyclyl where R^(c) is an alkynylene chain as defined above. The alkynylene chain and the carbocyclyl radical is optionally substituted as defined above.

“Carbocyclylalkoxy” refers to a radical bonded through an oxygen atom of the formula —O—R^(c)-carbocyclyl where R^(c) is an alkylene chain as defined above. The alkylene chain and the carbocyclyl radical is optionally substituted as defined above.

As used herein, “carboxylic acid bioisostere” refers to a functional group or moiety that exhibits similar physical, biological and/or chemical properties as a carboxylic acid moiety. Examples of carboxylic acid bioisosteres include, but are not limited to,

and the like.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo substituents.

“Fluoroalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more fluoro radicals, as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. In some embodiments, the alkyl part of the fluoroalkyl radical is optionally substituted as defined above for an alkyl group.

“Heterocyclyl” refers to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes fused or bridged ring systems. The heteroatoms in the heterocyclyl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl radical is partially or fully saturated. The heterocyclyl is attached to the rest of the molecule through any atom of the ring(s). Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, the term “heterocyclyl” is meant to include heterocyclyl radicals as defined above that are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each R^(b) is independently a direct bond or a straight or branched alkylene or alkenylene chain, and R^(c) is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

“N-heterocyclyl” or “N-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. An N-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such N-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

“C-heterocyclyl” or “C-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one heteroatom and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a carbon atom in the heterocyclyl radical. A C-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such C-heterocyclyl radicals include, but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.

“Heterocyclylalkyl” refers to a radical of the formula —R^(c)-heterocyclyl where R^(c) is an alkylene chain as defined above. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heterocyclylalkyl radical is optionally substituted as defined above for an alkylene chain. The heterocyclyl part of the heterocyclylalkyl radical is optionally substituted as defined above for a heterocyclyl group.

“Heterocyclylalkoxy” refers to a radical bonded through an oxygen atom of the formula —O—R^(c)-heterocyclyl where R^(c) is an alkylene chain as defined above. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heterocyclylalkoxy radical is optionally substituted as defined above for an alkylene chain. The heterocyclyl part of the heterocyclylalkoxy radical is optionally substituted as defined above for a heterocyclyl group.

“Heteroaryl” refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, the heteroaryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system in accordance with the Hickel theory. Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in the specification, the term “heteroaryl” is meant to include heteroaryl radicals as defined above which are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each R^(b) is independently a direct bond or a straight or branched alkylene or alkenylene chain, and R^(c) is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

“N-heteroaryl” refers to a heteroaryl radical as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogen atom in the heteroaryl radical. An N-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

“C-heteroaryl” refers to a heteroaryl radical as defined above and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a carbon atom in the heteroaryl radical. A C-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

“Heteroarylalkyl” refers to a radical of the formula —R^(c)-heteroaryl, where R^(c) is an alkylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heteroarylalkyl radical is optionally substituted as defined above for an alkylene chain. The heteroaryl part of the heteroarylalkyl radical is optionally substituted as defined above for a heteroaryl group.

“Heteroarylalkoxy” refers to a radical bonded through an oxygen atom of the formula —O—R^(c)-heteroaryl, where R^(c) is an alkylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heteroarylalkoxy radical is optionally substituted as defined above for an alkylene chain. The heteroaryl part of the heteroarylalkoxy radical is optionally substituted as defined above for a heteroaryl group.

The compounds disclosed herein, in some embodiments, contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R)- or (S)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the compounds disclosed herein are contemplated by this disclosure. When the compounds described herein contain alkene double bonds, and unless specified otherwise, it is intended that this disclosure includes both E and Z geometric isomers (e.g., cis or trans.) Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term “geometric isomer” refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term “positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para-isomers around a benzene ring.

A “tautomer” refers to a molecule wherein a proton shift from one atom of a molecule to another atom of the same molecule is possible. The compounds presented herein, in certain embodiments, exist as tautomers. In circumstances where tautomerization is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors, including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium include:

The compounds disclosed herein, in some embodiments, are used in different enriched isotopic forms, e.g., enriched in the content of ²H, ³H, ¹¹C, ¹³C and/or ¹⁴C. In one particular embodiment, the compound is deuterated in at least one position. Such deuterated forms can be made by the procedure described in U.S. Pat. Nos. 5,846,514 and 6,334,997. As described in U.S. Pat. Nos. 5,846,514 and 6,334,997, deuteration can improve the metabolic stability and or efficacy, thus increasing the duration of action of drugs.

Unless otherwise stated, structures depicted herein are intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are within the scope of the present disclosure.

The compounds of the present disclosure optionally contain unnatural proportions of atomic isotopes at one or more atoms that constitute such compounds. For example, the compounds may be labeled with isotopes, such as for example, deuterium (²H), tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). Isotopic substitution with ²H, ¹¹C, ¹³C, ¹⁴C, ¹⁵C, ¹²N, ¹³N, ¹⁵N, ¹⁶N, ¹⁶O, ¹⁷O, ¹⁴F, ¹⁵F, ¹⁶F, ¹⁷F, ¹⁸F, ³³S, ³⁴S, ³⁵S, ³⁶S, ³⁵Cl, ³⁷Cl, ⁷⁹Br, ⁸¹Br, ¹²⁵I are all contemplated. All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

In certain embodiments, the compounds disclosed herein have some or all of the ¹H atoms replaced with ²H atoms. The methods of synthesis for deuterium-containing compounds are known in the art and include, by way of non-limiting example only, the following synthetic methods.

Deuterium substituted compounds are synthesized using various methods such as described in: Dean, Dennis C.; Editor. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [In: Curr., Pharm. Des., 2000; 6(10)] 2000, 110 pp; George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.

Deuterated starting materials are readily available and are subjected to the synthetic methods described herein to provide for the synthesis of deuterium-containing compounds. Large numbers of deuterium-containing reagents and building blocks are available commercially from chemical vendors, such as Aldrich Chemical Co.

Deuterium-transfer reagents suitable for use in nucleophilic substitution reactions, such as iodomethane-d₃ (CD₃I), are readily available and may be employed to transfer a deuterium-substituted carbon atom under nucleophilic substitution reaction conditions to the reaction substrate. The use of CD₃I is illustrated, by way of example only, in the reaction schemes below.

Deuterium-transfer reagents, such as lithium aluminum deuteride (LiAlD₄), are employed to transfer deuterium under reducing conditions to the reaction substrate. The use of LiAlD₄ is illustrated, by way of example only, in the reaction schemes below.

Deuterium gas and palladium catalyst are employed to reduce unsaturated carbon-carbon linkages and to perform a reductive substitution of aryl carbon-halogen bonds as illustrated, by way of example only, in the reaction schemes below.

In one embodiment, the compounds disclosed herein contain one deuterium atom. In another embodiment, the compounds disclosed herein contain two deuterium atoms. In another embodiment, the compounds disclosed herein contain three deuterium atoms. In another embodiment, the compounds disclosed herein contain four deuterium atoms. In another embodiment, the compounds disclosed herein contain five deuterium atoms. In another embodiment, the compounds disclosed herein contain six deuterium atoms. In another embodiment, the compounds disclosed herein contain more than six deuterium atoms. In another embodiment, the compound disclosed herein is fully substituted with deuterium atoms and contains no non-exchangeable ¹H hydrogen atoms. In one embodiment, the level of deuterium incorporation is determined by synthetic methods in which a deuterated synthetic building block is used as a starting material.

“Pharmaceutically acceptable salt” includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the opioid receptor antagonist prodrug compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 66:1-19 (1997)). Acid addition salts of basic compounds are, in some embodiments, prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

“Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts are, in some embodiments, formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.

As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient is still afflicted with the underlying disorder. For prophylactic benefit, the compositions are, in some embodiments, administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease has not been made.

Opioid Receptor Pharmacology

The opioid receptors, μ, δ, κ, and the opioid-like receptor ORL-1 belong to the super family of G-protein coupled receptors (GPCRs) that possess seven helical trans-membrane spanning domains in their architecture. The majority of research efforts focused upon this group of proteins has been directed toward the μ receptor since it mediates the actions of both the opiate and opioid analgesics such as morphine and fentanyl, respectively. However, over the years it has become increasingly clear that the entire family of proteins is actively involved in a host of biological processes. Furthermore, the advent of selective antagonists has demonstrated that pharmacotherapeutic opportunities exist via both negative and positive modulation of this receptor family.

The μ (mu, OP₃ or MOP) receptor was originally defined and characterized pharmacologically by Martin, Kosterlitz and their colleagues on the basis of its high affinity for, and sensitivity to, morphine (Martin et al. The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog J. Pharmacol. Exp. Ther. (1976), 197: 517-532; Kosterlitz, et al. Endogenous opioid peptides: multiple agonists and receptors, Nature (1977) 267: 495-499). The endogenous opioids, [Met⁵]-enkephalin, [Leu⁵]-enkephalin, extended forms of [Met⁵]-enkephalin including metorphamide and BAM-18, β-endorphin, and truncated forms of dynorphin (e.g. dynorphin-(1-9) and shorter dynorphin peptides), also have affinities for receptors that are consistent with a possible role for each of these peptides as natural ligands for this receptor type, although these endogenous peptides are not selective for receptors. Two putative natural ligands, endomorphin-1 and -2, that appear to mediate their effects exclusively through the opioid receptor, also have been reported to be present in brain although no gene, precursor protein, or other mechanism for their endogenous synthesis has been identified.

The receptors are distributed throughout the neuraxis. The highest μ receptor densities are found in the thalamus, caudate putamen, neocortex, nucleus accumbens, amygdala, interpeduncular complex, and inferior and superior colliculi (Watson et al. Autoradiographic differentiation of mu, delta and kappa receptors in the rat forebrain and midbrain, J. Neurosci. (1987), 7: 2445-2464). The receptors, as well as δ and κ receptors, are also present in the superficial layers of the dorsal horn of spinal cord. A moderate density of μ receptors is found in periaqueductal gray and raphé nuclei. These brain regions have a well-established role in pain and analgesia. Other physiological functions regulated by receptors include respiratory and cardiovascular functions, intestinal transit, feeding, mood, thermoregulation, hormone secretion and immune functions.

The δ (delta, OP₁ or DOP) opioid receptor was defined using the mouse vas deferens preparation and the enkephalins are generally considered the preferred endogenous ligands. The δ receptors are discretely distributed in the central nervous system (CNS), with a prominent gradient of receptor density from high levels in forebrain structures to relatively low levels in most hindbrain regions. The highest densities are found in olfactory bulb, neocortex, caudate putamen, nucleus accumbens, and amygdala (Watson et al. Autoradiographic differentiation of mu, delta and kappa receptors in the rat forebrain and midbrain, J. Neurosci. (1987), 7: 2445-2464). The thalamus and hypothalamus have a moderate density of 6 receptors; in more caudal regions the interpeduncular nucleus and pontine nuclei show high binding in rat, but much lower levels in mouse (Kitchen et al. Quantitative autoradiographic mapping of mu, delta and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene, Brain Res. (1997), 778: 73-88). In the spinal cord, δ receptors are present in dorsal horn where they play a role in mediating the analgesic effects of δ agonists.

The κ (kappa, OP₂ or KOP) opioid receptor was first proposed on the basis of in vivo studies in dogs with ketocyclazocine and related drugs (Martin et al. The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog J. Pharmacol. Exp. Ther. (1976), 197: 517-532). Subsequent studies have confirmed the presence of this receptor type in other species including guinea pig, a species that was preferred for many of the early studies on kappa opioid receptors. Dynorphins A and B and α-neoendorphin appear to be the endogenous ligands for opioid κ receptors, although shorter peptides derived from prodynorphin have comparable affinities at μ and κ receptors. The κ receptors are located predominantly in the cerebral cortex, nucleus accumbens, claustrum and hypothalamus of rat and mouse (Kitchen et al. Quantitative autoradiographic mapping of mu, delta and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene, Brain Res. (1997), 778: 73-88; Watson et al. Autoradiographic differentiation of mu, delta and kappa receptors in the rat forebrain and midbrain, J. Neurosci. (1987), 7: 2445-2464), and have been implicated in the regulation of nociception, diuresis, feeding, neuroendocrine and immune system functions (Dhawan et al. International Union of Pharmacology. XII. Classification for opioid receptors, Pharmacol. Rev. (1996), 48: 567-592).

ORL1 receptors (also called nociceptin, or orphaninFQ receptors) are the youngest members of the opioid receptor family. Agonist-induced internalization of ORL1 is rapid and concentration dependent. Agonist challenge also reduces the ability of ORL1 to couple to inhibition of forskolin-stimulated cAMP production, suggesting that ORL1 undergoes similar desensitization mechanisms as compared with the other three opioid receptors subtypes.

The structure of the ORL1 receptor indicates that it has evolved as part of the opioid receptor family. Sequence comparisons with μ, κ, and δ receptors, and with other similar G protein-coupled receptors (e.g. of the SOM receptor family), indicate that the ORL1 receptor is more closely related to opioid receptors than to other types of G protein-coupled receptors (Birgul, et al. Reverse Physiology in drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family. EMBO J. (1999), 18: 5892-5900). Additionally, agonists at ORL1 receptors induce activation of the same set of transduction pathways activated by μ, κ, and δ receptors, and the endogenous ligand, ORL1, shares considerable sequence homology with dynorphin A and, to a lesser extent, with the enkephalins. Thus, the ORL1 receptor and its endogenous ligand are closely related in an evolutionary sense to the μ, κ, and δ receptors.

Despite the evidence of evolutionary and functional homology, the ORL1 receptor is not an opioid receptor from a pharmacological perspective. The effects of activation of this receptor are not obviously ‘opiate-like’ with respect to pain perception. The ORL1 receptor has negligible affinity for naloxone and for most other antagonists at μ, κ or δ receptors. The ORL1 receptor is, however, expressed in many functional systems in which endogenous opioids play a regulatory role. Although the functions of ORL1 are not yet fully understood, regulatory functions for ORL1 parallel to but not identical to those of the endogenous opioid peptides seem very probable. Despite these functional differences, the subcommittee finds the structural relationship between the ORL1 receptor and μ, δ and κ receptors compelling.

ORL1 receptor regulation, while increasingly studied, is still in the infant stages of understanding when compared to the other three opioid receptor subtypes. To date few site-directed mutagenesis studies have been conducted, and receptor regulation in primary neurons, dorsal root ganglion, or dorsal horn neurons remains unknown.

An integral part of the effort to characterize the opioid receptor system has been the discovery of potent, pure antagonists of opioid receptors. Nalmefene (1a) and naltrexone (1b), both competitive antagonists at μ, δ, and κ opioid receptors, were used as pharmacological tools to identify and characterize opioid systems.

Nalmefene is an opioid receptor antagonist that has been available for several years as Revex® injection for use in reversing opioid effects and for opioid overdose. Nalmefene is also described in literature for the treatment of substance abuse disorders such as alcohol dependence and abuse, and impulse control disorders such as pathological gambling and addiction to shopping. It is marketed as Selincro in Europe as an on demand oral pill for alcohol abuse. It has the IUPAC name 17-cyclopropylmethyl-4,5α-epoxy-6-methylenemorphinan-3,14-diol and has the structure provided in Formula (1A).

Naltrexone is an opioid receptor antagonist used primarily in the management of alcohol dependence and opioid dependence. It is marketed in the generic form as its hydrochloride salt, naltrexone hydrochloride under the trade names Revia® and Depade® in the form of 50 mg film coated tablets. Once monthly extended release naltrexone, marketed in the United States as Vivitrol, has gained wide acceptance in opioid use disorder due to increased patient adherence. Naltrexone has the IUPAC name 17-(cyclopropylmethyl)-4,5α-epoxy-3,14-dihydroxymorphinan-6-one and has the structure provided in Formula (1B)

Low doses of naltrexone have also been investigated in patients with multiple sclerosis, autism, active Crohn's disease, AIDS, rheumatoid arthritis, celiac disease, certain forms of cancer, and autoimmune diseases. Opioids act as cytokines, the principal communication signallers of the immune system, creating immunomodulatory effects through opioid receptors on immune cells. Very low doses of naltrexone were shown to boost the immune system and helps to fight against diseases characterized by inadequate immune function.

In terms of pharmacology, naltrexone blocks the effects of opioids by its highly competitive binding at the μ-opioid receptors. Being a competitive antagonist, the suppression of an opiate's agonistic, euphorigenic effect can be overcome. However, clinical studies have indicated that naltrexone in an oral dosage of approximately 50 mg is able to block the pharmacological effects of up to 25 mg of intravenously administered heroin for periods as long as twenty four hours.

The mechanism of action of naltrexone in the treatment of alcoholism is not understood although involvement of the endogenous opioid system is suggested by preclinical data. Opioid antagonists have been shown to reduce alcohol consumption by animals, and naltrexone has shown efficacy in maintaining abstinence in clinical studies in humans.

Opioid Receptor Antagonists Prodrugs

Although using nalmefene and naltrexone in the treatment of alcohol dependence and opioid dependence provides a great benefit to the society, the problem with these drugs is that they have very short period of action. Thus, for example, well absorbed orally (approximately 96% of an oral dose is absorbed from the gastrointestinal tract), naltrexone is subject to significant first pass metabolism with oral bioavailability estimates ranging from 5% to 40%. The activity of naltrexone is believed to be as a result of both naltrexone and its 6-β-naltrexol metabolite. Two other minor metabolites are 2-hydroxy-3-methoxy-6-β-naltrexol and 2-hydroxy-3-methyl-naltrexone. Peak plasma levels of both naltrexone and 6-β-naltexol occur within one hour after oral dosing; mean elimination half-life values for naltrexone and 6-β-naltrexol are four and thirteen hours respectively. Even for long acting naltrexone injections, clinicians indicate that patients discontinue treatment too early. Therefore, a need exists for ultra-long acting opioid antagonists in the treatment of substance abuse disorder.

One of the solutions to overcome the problem of short period of action of nalmefene and naltrexone is to use prodrugs which provide a long, sustained, and controlled release of nalmefene and naltrexone opioid receptor antagonists upon administration into the body.

As used in this disclosure, the term “prodrug” is meant to indicate a compound that is converted under physiological conditions to nalmefene or naltrexone. A prodrug, in some embodiments, is inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis. Thus, the term “prodrug” refers to a precursor compound that is pharmaceutically acceptable, and in some embodiments, is devoid of the pharmacological properties of nalmefene or naltrexone. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).

A discussion of prodrugs is provided in Higuchi, T., et al., “Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of nalmefene or naltrexone, as described herein, are prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved to the parent active compound. Prodrugs include compounds wherein a hydroxy group is bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, cleaves to form a free hydroxy group.

Provided herein are prodrugs of opioid receptor antagonists nalmefene and naltrexone.

In one aspect, provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (I),

wherein,

X is O or CH₂;

R is selected from:

a. (C₃-C₇cycloalkyl)CH₂C(O)—;

b. (C₃-C₇cycloalkyl)CH₂CH₂C(O)—;

c. —C(O)OC₇-C₂₀ alkyl; or

d. —C(O)NHC(CH₃)₃.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R is (C₃-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₃-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is —C(O)OC₇-C₂₀ alkyl. In some embodiments, R is —C(O)NHC(CH₃)₃.

In some embodiments, R is (C₃-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₃-C₄cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₃-C₅cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₃-C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₄-C₅cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₄-C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₄-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₅-C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₅-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₆-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₃cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₄cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₅cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is (C₇cycloalkyl)CH₂C(O)—.

In some embodiments, R is (C₃-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₃-C₄cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₃-C₅cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₃-C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₄-C₅cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₄-C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₄-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₅-C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₅-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₆-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₃cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₄cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₅cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is (C₇cycloalkyl)CH₂CH₂C(O)—.

In some embodiments, R is —C(O)OC₇-C₂₀ alkyl. In some embodiments, R is —C(O)OC₇-C₅ alkyl. In some embodiments, R is —C(O)OC₇-C₉ alkyl. In some embodiments, R is —C(O)OC₇-C₁₀ alkyl. In some embodiments, R is —C(O)OC₇-C₁₁ alkyl. In some embodiments, R is —C(O)OC₇-C₁₂ alkyl. In some embodiments, R is —C(O)OC₇-C₁₃ alkyl. In some embodiments, R is —C(O)OC₇-C₁₄ alkyl. In some embodiments, R is —C(O)OC₇-C₁₅ alkyl. In some embodiments, R is —C(O)OC₇-C₁₆ alkyl. In some embodiments, R is —C(O)OC₇-C₁₇ alkyl. In some embodiments, R is —C(O)OC₇-C₁₈ alkyl. In some embodiments, R is —C(O)OC₇-C₁₉ alkyl. In some embodiments, R is —C(O)OC₈-C₉ alkyl. In some embodiments, R is —C(O)OC₈-C₁₀ alkyl. In some embodiments, R is —C(O)OC₈-C₁₁ alkyl. In some embodiments, R is —C(O)OC₈-C₁₂ alkyl. In some embodiments, R is —C(O)OC₈-C₁₃ alkyl. In some embodiments, R is —C(O)OC₈-C₁₄ alkyl. In some embodiments, R is —C(O)OC₈-C₁₅ alkyl. In some embodiments, R is —C(O)OC₈-C₁₆ alkyl. In some embodiments, R is —C(O)OC₈-C₁₇ alkyl. In some embodiments, R is —C(O)OC₈-C₁₈ alkyl. In some embodiments, R is —C(O)OC₈-C₁₉ alkyl. In some embodiments, R is —C(O)OC₈-C₂₀ alkyl. In some embodiments, R is —C(O)OC₉-C₁₀ alkyl. In some embodiments, R is —C(O)OC₉-C₁₁ alkyl. In some embodiments, R is —C(O)OC₉-C₁₂ alkyl. In some embodiments, R is —C(O)OC₉-C₁₃ alkyl. In some embodiments, R is —C(O)OC₉-C₁₄ alkyl. In some embodiments, R is —C(O)OC₉-C₁₅ alkyl. In some embodiments, R is —C(O)OC₉-C₁₆ alkyl. In some embodiments, R is —C(O)OC₉₋C₁₇ alkyl. In some embodiments, R is —C(O)OC₉-C₁₈ alkyl. In some embodiments, R is —C(O)OC₉₋C₁₉ alkyl. In some embodiments, R is —C(O)OC₉-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₁ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₂ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₃ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₄ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₇ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₀-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₂ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₃ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₄ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₇ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₁-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₃ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₄ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₇ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₂-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₄ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₇ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₃-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₄-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₄-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₄-C₁₇ alkyl. In some embodiments, R is —C(O)OC₁₄-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₄-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₄-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₅-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₅-C₁₇ alkyl. In some embodiments, R is —C(O)OC₁₅-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₅-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₅-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₆-C₁₇ alkyl. In some embodiments, R is —C(O)OC₁₆₋C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₆-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₆-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₇-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₇-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₇-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₈-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₈-C₂₀ alkyl. In some embodiments, R is —C(O)OC₁₉-C₂₀ alkyl. In some embodiments, R is —C(O)OC₇ alkyl. In some embodiments, R is —C(O)OC₈ alkyl. In some embodiments, R is —C(O)OC₉ alkyl. In some embodiments, R is —C(O)OC₁₀ alkyl. In some embodiments, R is —C(O)OC₁₁ alkyl. In some embodiments, R is —C(O)OC₁₂ alkyl. In some embodiments, R is —C(O)OC₁₃ alkyl. In some embodiments, R is —C(O)OC₁₄ alkyl. In some embodiments, R is —C(O)OC₁₅ alkyl. In some embodiments, R is —C(O)OC₁₆ alkyl. In some embodiments, R is —C(O)OC₁₇ alkyl. In some embodiments, R is —C(O)OC₁₈ alkyl. In some embodiments, R is —C(O)OC₁₉ alkyl. In some embodiments, R is —C(O)OC₂₀ alkyl.

In some embodiments, R is —C(O)NHC(CH₃)₃.

In another aspect, also provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (II),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 7-15; provided if X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl.

In some embodiments, R¹ is a C₄-C₁₀ alkyl. In some embodiments, R¹ is a C₄-C₅ alkyl. In some embodiments, R¹ is a C₄-C₆ alkyl. In some embodiments, R¹ is a C₄-C₇ alkyl. In some embodiments, R¹ is a C₄-C₈ alkyl. In some embodiments, R¹ is a C₄-C₉ alkyl. In some embodiments, R¹ is a C₅-C₆ alkyl. In some embodiments, R¹ is a C₅-C₇ alkyl. In some embodiments, R¹ is a C₅-C₈ alkyl. In some embodiments, R¹ is a C₅-C₉ alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments, R¹ is a C₆-C₇ alkyl. In some embodiments, R¹ is a C₆-C₈ alkyl. In some embodiments, R¹ is a C₆-C₉ alkyl. In some embodiments, R¹ is a C₆-C₁₀ alkyl. In some embodiments, R¹ is a C₇-C₈ alkyl. In some embodiments, R¹ is a C₇-C₉ alkyl. In some embodiments, R¹ is a C₇-C₁₀ alkyl. In some embodiments, R¹ is a C₈-C₉ alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments, R¹ is a C₉-C₁₀ alkyl. In some embodiments, R¹ is a C₄ alkyl. In some embodiments, R¹ is a C₅ alkyl. In some embodiments, R¹ is a C₆ alkyl. In some embodiments, R¹ is a C₇ alkyl. In some embodiments, R¹ is a C₈ alkyl. In some embodiments, R¹ is a C₉ alkyl. In some embodiments, R¹ is a C₁₀ alkyl.

In some embodiments, R¹ is a C₄-C₁₀ alkenyl. In some embodiments, R¹ is a C₄-C₅ alkenyl. In some embodiments, R¹ is a C₄-C₆ alkenyl. In some embodiments, R¹ is a C₄-C₇ alkenyl. In some embodiments, R¹ is a C₄-C₈ alkenyl. In some embodiments, R¹ is a C₄-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₆ alkenyl. In some embodiments, R¹ is a C₅-C₇ alkenyl. In some embodiments, R¹ is a C₅-C₈ alkenyl. In some embodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₁₀ alkenyl. In some embodiments, R¹ is a C₆-C₇ alkenyl. In some embodiments, R¹ is a C₆-C₈ alkenyl. In some embodiments, R¹ is a C₆-C₉ alkenyl. In some embodiments, R¹ is a C₆-C₁₀ alkenyl. In some embodiments, R¹ is a C₇-C₈ alkenyl. In some embodiments, R¹ is a C₇-C₉ alkenyl. In some embodiments, R¹ is a C₇-C₁₀ alkenyl. In some embodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₈-C₁₀ alkenyl. In some embodiments, R¹ is a C₉-C₁₀ alkenyl. In some embodiments, R¹ is a C₄ alkenyl. In some embodiments, R¹ is a C₅ alkenyl. In some embodiments, R¹ is a C₆ alkenyl. In some embodiments, R¹ is a C₇ alkenyl. In some embodiments, R¹ is a C₈ alkenyl. In some embodiments, R¹ is a C₉ alkenyl. In some embodiments, R¹ is a C₁₀ alkenyl.

In some embodiments, n is 7-15. In some embodiments, n is 7-8. In some embodiments, n is 7-9. In some embodiments, n is 7-10. In some embodiments, n is 7-11. In some embodiments, n is 7-12. In some embodiments, n is 7-13. In some embodiments, n is 7-14. In some embodiments, n is 8-9. In some embodiments, n is 8-10. In some embodiments, n is 8-11. In some embodiments, n is 8-12. In some embodiments, n is 8-13. In some embodiments, n is 8-14. In some embodiments, n is 8-15. In some embodiments, n is 9-10. In some embodiments, n is 9-11. In some embodiments, n is 9-12. In some embodiments, n is 9-13. In some embodiments, n is 9-14. In some embodiments, n is 9-15. In some embodiments, n is 10-11. In some embodiments, n is 10-12. In some embodiments, n is 10-13. In some embodiments, n is 10-14. In some embodiments, n is 10-15. In some embodiments, n is 11-12. In some embodiments, n is 11-13. In some embodiments, n is 11-14. In some embodiments, n is 11-15. In some embodiments, n is 12-13. In some embodiments, n is 12-14. In some embodiments, n is 12-15. In some embodiments, n is 13-14. In some embodiments, n is 13-15. In some embodiments, n is 14-15. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is 11. In some embodiments, n is 12. In some embodiments, n is 13. In some embodiments, n is 14. In some embodiments, n is 15.

In another aspect, also provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (II),

wherein,

X is O or CH₂;

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 7-15; provided if X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl.

In some embodiments, R¹ is a C₄-C₁₀ alkyl. In some embodiments, R¹ is a C₄-C₅ alkyl. In some embodiments, R¹ is a C₄-C₆ alkyl. In some embodiments, R¹ is a C₄-C₇ alkyl. In some embodiments, R¹ is a C₄-C₈ alkyl. In some embodiments, R¹ is a C₄-C₉ alkyl. In some embodiments, R¹ is a C₅-C₆ alkyl. In some embodiments, R¹ is a C₅-C₇ alkyl. In some embodiments, R¹ is a C₅-C₈ alkyl. In some embodiments, R¹ is a C₅-C₉ alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments, R¹ is a C₆-C₇ alkyl. In some embodiments, R¹ is a C₆-C₈ alkyl. In some embodiments, R¹ is a C₆-C₉ alkyl. In some embodiments, R¹ is a C₆-C₁₀ alkyl. In some embodiments, R¹ is a C₇-C₈ alkyl. In some embodiments, R¹ is a C₇-C₉ alkyl. In some embodiments, R¹ is a C₇-C₁₀ alkyl. In some embodiments, R¹ is a C₅-C₉ alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments, R¹ is a C₉-C₁₀ alkyl. In some embodiments, R¹ is a C₄ alkyl. In some embodiments, R¹ is a C₅ alkyl. In some embodiments, R¹ is a C₆ alkyl. In some embodiments, R¹ is a C₇ alkyl. In some embodiments, R¹ is a C₈ alkyl. In some embodiments, R¹ is a C₉ alkyl. In some embodiments, R¹ is a C₁₀ alkyl.

In some embodiments, R¹ is a C₄-C₁₀ alkenyl. In some embodiments, R¹ is a C₄-C₅ alkenyl. In some embodiments, R¹ is a C₄-C₆ alkenyl. In some embodiments, R¹ is a C₄-C₇ alkenyl. In some embodiments, R¹ is a C₄-C₈ alkenyl. In some embodiments, R¹ is a C₄-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₆ alkenyl. In some embodiments, R¹ is a C₅-C₇ alkenyl. In some embodiments, R¹ is a C₅-C₈ alkenyl. In some embodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₁₀ alkenyl. In some embodiments, R¹ is a C₆-C₇ alkenyl. In some embodiments, R¹ is a C₆-C₈ alkenyl. In some embodiments, R¹ is a C₆-C₉ alkenyl. In some embodiments, R¹ is a C₆-C₁₀ alkenyl. In some embodiments, R¹ is a C₇-C₈ alkenyl. In some embodiments, R¹ is a C₇-C₉ alkenyl. In some embodiments, R¹ is a C₇-C₁₀ alkenyl. In some embodiments, R¹ is a C₈-C₉ alkenyl. In some embodiments, R¹ is a C₈-C₁₀ alkenyl. In some embodiments, R¹ is a C₉-C₁₀ alkenyl. In some embodiments, R¹ is a C₄ alkenyl. In some embodiments, R¹ is a C₅ alkenyl. In some embodiments, R¹ is a C₆ alkenyl. In some embodiments, R¹ is a C₇ alkenyl. In some embodiments, R¹ is a C₈ alkenyl. In some embodiments, R¹ is a C₉ alkenyl. In some embodiments, R¹ is a C₁₀ alkenyl.

In some embodiments, n is 7-15. In some embodiments, n is 7-8. In some embodiments, n is 7-9. In some embodiments, n is 7-10. In some embodiments, n is 7-11. In some embodiments, n is 7-12. In some embodiments, n is 7-13. In some embodiments, n is 7-14. In some embodiments, n is 8-9. In some embodiments, n is 8-10. In some embodiments, n is 8-11. In some embodiments, n is 8-12. In some embodiments, n is 8-13. In some embodiments, n is 8-14. In some embodiments, n is 8-15. In some embodiments, n is 9-10. In some embodiments, n is 9-11. In some embodiments, n is 9-12. In some embodiments, n is 9-13. In some embodiments, n is 9-14. In some embodiments, n is 9-15. In some embodiments, n is 10-11. In some embodiments, n is 10-12. In some embodiments, n is 10-13. In some embodiments, n is 10-14. In some embodiments, n is 10-15. In some embodiments, n is 11-12. In some embodiments, n is 11-13. In some embodiments, n is 11-14. In some embodiments, n is 11-15. In some embodiments, n is 12-13. In some embodiments, n is 12-14. In some embodiments, n is 12-15. In some embodiments, n is 13-14. In some embodiments, n is 13-15. In some embodiments, n is 14-15. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is 11. In some embodiments, n is 12. In some embodiments, n is 13. In some embodiments, n is 14. In some embodiments, n is 15.

In another aspect, also provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (IIa),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 9-15.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl.

In some embodiments, R¹ is a C₄-C₁₀ alkyl. In some embodiments, R¹ is a C₄-C₅ alkyl. In some embodiments, R¹ is a C₄-C₆ alkyl. In some embodiments, R¹ is a C₄-C₇ alkyl. In some embodiments, R¹ is a C₄-C₈ alkyl. In some embodiments, R¹ is a C₄-C₉ alkyl. In some embodiments, R¹ is a C₅-C₆ alkyl. In some embodiments, R¹ is a C₅-C₇ alkyl. In some embodiments, R¹ is a C₅-C₈ alkyl. In some embodiments, R¹ is a C₅-C₉ alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments, R¹ is a C₆-C₇ alkyl. In some embodiments, R¹ is a C₆-C₈ alkyl. In some embodiments, R¹ is a C₆-C₉ alkyl. In some embodiments, R¹ is a C₆-C₁₀ alkyl. In some embodiments, R¹ is a C₇-C₈ alkyl. In some embodiments, R¹ is a C₇-C₉ alkyl. In some embodiments, R¹ is a C₇-C₁₀ alkyl. In some embodiments, R¹ is a C₅-C₉ alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments, R¹ is a C₉-C₁₀ alkyl. In some embodiments, R¹ is a C₄ alkyl. In some embodiments, R¹ is a C₅ alkyl. In some embodiments, R¹ is a C₆ alkyl. In some embodiments, R¹ is a C₇ alkyl. In some embodiments, R¹ is a C₈ alkyl. In some embodiments, R¹ is a C₉ alkyl. In some embodiments, R¹ is a C₁₀ alkyl.

In some embodiments, R¹ is a C₄-C₁₀ alkenyl. In some embodiments, R¹ is a C₄-C₅ alkenyl. In some embodiments, R¹ is a C₄-C₆ alkenyl. In some embodiments, R¹ is a C₄-C₇ alkenyl. In some embodiments, R¹ is a C₄-C₈ alkenyl. In some embodiments, R¹ is a C₄-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₆ alkenyl. In some embodiments, R¹ is a C₅-C₇ alkenyl. In some embodiments, R¹ is a C₅-C₈ alkenyl. In some embodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₁₀ alkenyl. In some embodiments, R¹ is a C₆-C₇ alkenyl. In some embodiments, R¹ is a C₆-C₈ alkenyl. In some embodiments, R¹ is a C₆-C₉ alkenyl. In some embodiments, R¹ is a C₆-C₁₀ alkenyl. In some embodiments, R¹ is a C₇-C₈ alkenyl. In some embodiments, R¹ is a C₇-C₉ alkenyl. In some embodiments, R¹ is a C₇-C₁₀ alkenyl. In some embodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₈-C₁₀ alkenyl. In some embodiments, R¹ is a C₉-C₁₀ alkenyl. In some embodiments, R¹ is a C₄ alkenyl. In some embodiments, R¹ is a C₅ alkenyl. In some embodiments, R¹ is a C₆ alkenyl. In some embodiments, R¹ is a C₇ alkenyl. In some embodiments, R¹ is a C₈ alkenyl. In some embodiments, R¹ is a C₉ alkenyl. In some embodiments, R¹ is a C₁₀ alkenyl.

In some embodiments, n is 9-15. In some embodiments, n is 9-10. In some embodiments, n is 9-11. In some embodiments, n is 9-12. In some embodiments, n is 9-13. In some embodiments, n is 9-14. In some embodiments, n is 10-11. In some embodiments, n is 10-12. In some embodiments, n is 10-13. In some embodiments, n is 10-14. In some embodiments, n is 10-15. In some embodiments, n is 11-12. In some embodiments, n is 11-13. In some embodiments, n is 11-14. In some embodiments, n is 11-15. In some embodiments, n is 12-13. In some embodiments, n is 12-14. In some embodiments, n is 12-15. In some embodiments, n is 13-14. In some embodiments, n is 13-15. In some embodiments, n is 14-15. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is 11. In some embodiments, n is 12. In some embodiments, n is 13. In some embodiments, n is 14. In some embodiments, n is 15.

In another aspect, also provided herein is a compound, or pharmaceutically acceptable salt thereof, having a structure provided in Formula (III),

wherein,

X is O or CH₂;

R is selected from:

-   -   —[CH(R³)O]z-R⁴;     -   —[CH(R³)O]z-C(═O)OR⁴;     -   —[CH(R³)O]z-C(═O)NR⁴R⁵; and     -   —[CH(R³)O]z-P(═O)(OR⁴)(OR⁵);     -   wherein z is 1, 2, 3, 4, 5, 6, or 7;     -   R³ is hydrogen, halogen, alkyl, alkenyl, cycloalkylalkyl, or         aryl; each R⁴ and R⁵ is independently selected from hydrogen,         alkyl, alkenyl, cycloalkylalkyl,

or aryl.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, z is 4. In some embodiments, z is 5. In some embodiments, z is 6. In some embodiments, z is 7. In some embodiments, z is 1 or 2. In some embodiments, z is 2 or 3. In some embodiments, z is 1, 2, or 3.

In some embodiments, R³ is hydrogen, halogen or alkyl. In some embodiments, R³ is alkyl. In some embodiments, R³ is hydrogen. In some embodiments, R³ is hydrogen, halogen, alkyl, cycloalkylalkyl, or aryl. In some embodiments, R³ is hydrogen, halogen, cycloalkylalkyl, or aryl. In some embodiments, R³ is halogen. In some embodiments, the halogen is fluorine.

In some embodiments, each R⁴ and R⁵ is independently selected from alkyl, or aryl. In some embodiments, each R⁴ and R⁵ is independently selected from alkyl. In some embodiments, each R⁴ and R⁵ is independently selected from hydrogen or alkyl. In some embodiments, the alkyl is C₁₀-C₁₈ alkyl. In some embodiments, the alkyl is C₅-C₉ alkyl. In some embodiments, the alkyl is C₁-C₄ alkyl. In some embodiments, the alkyl is C₉-C₁₃ alkyl. In some embodiments, the alkyl is C₁₀-C₁₂ alkyl. In some embodiments, the alkyl is C₁₀ alkyl. In some embodiments, the alkyl is C₁₁ alkyl. In some embodiments, the alkyl is C₁₂ alkyl.

In some embodiments, R is: —[CH(R³)O]z-R⁴. In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴. In some embodiments, R is: —[CH(R³)O]z-C(═O)NR⁴R⁵. In some embodiments, R is: —[CH(R³)O]z-P(═O)(OR⁴)(OR⁵). In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴, wherein R³ is hydrogen, and R⁴ is C₉-C₁₃ alkyl. In some embodiments, R is: —[CH(R³)O]z—C(═O)OR⁴, wherein R³ is hydrogen, and R⁴ is C₁₀-C₁₂ alkyl. In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴, wherein R³ is hydrogen, and R⁴ is C₁₀ alkyl. In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴, wherein R³ is hydrogen, and R⁴ is C₁₁ alkyl. In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴, wherein R³ is hydrogen, and R⁴ is C₁₂ alkyl.

In some embodiments, the opioid receptor antagonist prodrug compound described herein has a structure provided in Table 1.

TABLE 1

Chemical Synthesis Example R X Chemical Name  1

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl ((E)- octadec-9-en-1-yl) carbonate  2

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl undecyl carbonate  3

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl undecyl carbonate  4

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl undecyl carbonate  5

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl undecyl carbonate  6

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl dodecyl carbonate  7

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl ((E)- octadec-9-en-1-yl) carbonate  8

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (E)- octadec-9-enoate  9

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl (E)- octadec-9-enoate 10

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl decyl carbonate 11

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl dodecyl carbonate 12

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl stearate 13

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (Z)- docos-13-enoate 14

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl docosanoate 15

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (E)- octadec-9-enoate 16

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl icosanoate 17

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl octyl carbonate 18

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl decyl carbonate 19

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl hexadecyl carbonate 20

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (9Z,12Z,15Z)-octadeca- 9,12,15-trienoate 21

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl hexadecyl carbonate 22

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (Z)- docos-13-enoate 23

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl octyl carbonate 24

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl dodecyl carbonate 25

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl tetradecyl carbonate 26

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl (E)- octadec-9-enoate 27

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl tetradecyl carbonate 28

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl icosyl carbonate 29

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl dodecyl carbonate 30

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl tridecyl carbonate 31

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl tetradecyl carbonate 32

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl pentadecyl carbonate 33

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl octadecyl carbonate 34

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl hexadecyl carbonate 35

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl decyl carbonate 36

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl oleate 37

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (9Z,12Z)-octadeca-9,12- dienoate 38

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl 3,3- dimethylbutanoate 39

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl 3- cyclopentylpropanoate 40

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl tert- butylcarbamate 41

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl oleate 42

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl 3,3- dimethylbutanoate 43

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl 3- cyclopentylpropanoate 44

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl dodecanoate 45

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl tetradecanoate 46

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl hexadecanoate 47

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl hexadecyl carbonate 48

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl dodecanoate 49

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9- yl)oxy)methyl hexadecanoate

In some embodiments, the opioid receptor antagonist prodrug compound described herein has a structure provided in Table 2.

TABLE 2

Ex- ample R X Chemical Name 50

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl icosanoate 51

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl docosanoate 52

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (9Z,12Z)-octadeca- 9,12,15-trienoate 53

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (9Z,12Z)-octadeca- 9,12-di.enoate 54

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl stearate 55

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl palmitate 56

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl palmitate 57

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl tetradecanoate 58

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl pentadecanoate 59

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl dodecanoate 60

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl yl)oxy)methyl tetradecanoate 61 H CH₂ Nalmefene 62 H O Naltrexone

The compounds used in the reactions described herein are made according to organic synthesis techniques known to those skilled in this art, starting from commercially available chemicals and/or from compounds described in the chemical literature. “Commercially available chemicals” are obtained from standard commercial sources including Acros Organics (Pittsburgh, Pa.), Aldrich Chemical (Milwaukee, Wis., including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park, UK), Avocado Research (Lancashire, U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester, Pa.), Crescent Chemical Co. (Hauppauge, N.Y.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, N.Y.), Fisher Scientific Co. (Pittsburgh, Pa.), Fisons Chemicals (Leicestershire, UK), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Key Organics (Cornwall, U.K.), Lancaster Synthesis (Windham, N.H.), Maybridge Chemical Co. Ltd. (Cornwall, U.K.), Parish Chemical Co. (Orem, Utah), Pfaltz & Bauer, Inc. (Waterbury, Conn.), Polyorganix (Houston, Tex.), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen AG (Hanover, Germany), Spectrum Quality Product, Inc. (New Brunswick, N.J.), TCI America (Portland, Oreg.), Trans World Chemicals, Inc. (Rockville, Md.), and Wako Chemicals USA, Inc. (Richmond, Va.).

Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds described herein, or provide references to articles that describe the preparation, include for example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., New York; S. R. Sandler et al., “Organic Functional Group Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O. House, “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Additional suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds described herein, or provide references to articles that describe the preparation, include for example, Fuhrhop, J. and Penzlin G. “Organic Synthesis: Concepts, Methods, Starting Materials”, Second, Revised and Enlarged Edition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, R. V. “Organic Chemistry, An Intermediate Text” (1996) Oxford University Press, ISBN 0-19-509618-5; Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations” 2nd Edition (1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. “Advanced Organic Chemistry: Reactions, Mechanisms, and Structure” 4th Edition (1992) John Wiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor) “Modern Carbonyl Chemistry” (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. “Patai's 1992 Guide to the Chemistry of Functional Groups” (1992) Interscience ISBN: 0-471-93022-9; Solomons, T. W. G. “Organic Chemistry” 7th Edition (2000) John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J. C., “Intermediate Organic Chemistry” 2nd Edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; “Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann's Encyclopedia” (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8 volumes; “Organic Reactions” (1942-2000) John Wiley & Sons, in over 55 volumes; and “Chemistry of Functional Groups” John Wiley & Sons, in 73 volumes.

Specific and analogous reactants are optionally identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (contact the American Chemical Society, Washington, D.C. for more details). Chemicals that are known but not commercially available in catalogs are optionally prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services. A reference for the preparation and selection of pharmaceutical salts of the opioid receptor antagonist prodrug compounds described herein is P. H. Stahl & C. G. Wermuth “Handbook of Pharmaceutical Salts”, Verlag Helvetica Chimica Acta, Zurich, 2002.

Pharmaceutical Compositions

In certain embodiments, the opioid receptor antagonist prodrug compound as described herein is administered as a pure chemical. In other embodiments, the opioid receptor antagonist prodrug compound described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, Pa. (2005)).

Provided herein is a pharmaceutical composition comprising at least one opioid receptor antagonist prodrug compound, or a stereoisomer, pharmaceutically acceptable salt, hydrate, solvate, or N-oxide thereof, together with one or more pharmaceutically acceptable carriers.

The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.

One embodiment provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the opioid receptor antagonist prodrug compound as described by any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as unreacted intermediates or synthesis by-products that are created, for example, in one or more of the steps of a synthesis method.

Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. In some embodiments, suitable nontoxic solid carriers are used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. (See, e.g., Remington: The Science and Practice of Pharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, Pa. (2005)).

In some embodiments, the opioid receptor antagonist prodrug compound as described by any one of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, is formulated for administration by injection. In some instances, the injection formulation is an aqueous formulation. In some instances, the injection formulation is a non-aqueous formulation. In some instances, the injection formulation is an oil-based formulation, such as sesame oil, cottonseed oil, or the like.

The dose of the composition comprising at least one opioid receptor antagonist prodrug compound as described herein differ, depending upon the patient's (e.g., human) condition, that is, general health status, age, and other factors.

Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity. Optimal doses are generally determined using experimental models and/or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient.

Dosing and Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once per day, twice per day, three times per day, four times per day or more.

The pharmaceutical composition is administered daily, every day, every alternate day, two days a week, three days a week, four days a week, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or other greater or lesser intervening frequency; also, it could be dosed once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once yearly, or with greater or lesser than aforementioned interval frequency. The pharmaceutical composition is administered for at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

In the case wherein the patient's status does not improve, upon the physician's discretion the administration of the composition is given continuously; alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). In some instances, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, 365 days, or 366 days. The dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be adjusted, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

In some embodiments, the amount of given opioid receptor antagonist prodrug compound varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

In some embodiments, the amount of given opioid receptor antagonist prodrug compound will typically be in the range of about 0.02 mg to about 5000 mg per dose. (Note: all prodrug mass quantities are expressed in base moiety equivalents). In some embodiments, the amount of given opioid receptor antagonist prodrug compound is in the range of about 1 mg to about 5000 mg per dose. In some embodiments, the amount of given opioid receptor antagonist prodrug compound is in the range of about 10 mg to about 1600 mg per dose. The desired dose may conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

In some embodiments, the daily dosages appropriate for the opioid receptor antagonist prodrug compound described herein are from about 0.01 mg/kg to about 30 mg/kg. In one embodiment, the daily dosages are from about 0.1 mg/kg to about 165 mg/kg. An indicated daily dosage in the larger mammal, including, but not limited to, humans, is in the range from about 0.5 mg to about 1000 mg, conveniently administered in a single dose or in divided doses. Suitable unit dosage forms for intramuscular administration include from about 1 to about 5000 mg active ingredient. In one embodiment, the unit dosage is about 10 mg, about 50 mg, about, 100 mg, about 200 mg, about 500 mg, about 1000 mg, about 2000 mg, about 2500 mg, about 4000 mg, or about 5000 mg.

The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages may be altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

Treatment of Behavioral Disorders

In some embodiments, described herein is a method of treating one or more medical conditions in a subject in need thereof, comprising administering to the subject in need thereof an opioid receptor antagonist compound described herein.

In some embodiments, the medical condition is selected from the group comprising opioid dependence, alcohol dependence, drug addiction, polydrug addiction and pain.

In some embodiments, described herein is an opioid receptor antagonist compound for use in reduction of opioid consumption in a patient with opioid dependence.

In some embodiments, described herein is an opioid receptor antagonist compound for use in reduction of alcohol consumption in a patient with alcohol dependence, pathological gambling shopping addiction or other diseases of compulsive behavior.

Provided herein is a method of treating opioid dependence in a patient in need thereof comprising administering a pharmaceutical composition comprising a compound of Formula (I), (II), (IIa), or (III), or a compound disclosed in Table 1, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Provided herein is the method wherein the pharmaceutical composition is administered orally. Provided herein is the method wherein the pharmaceutical composition is administered by injection. Provided herein is the method wherein the pharmaceutical composition is administered by intramuscular injection. Provided herein is the method wherein the intramuscular injection is a depot injection. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of 2 days to 3 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 2 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 4 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 7 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 10 days. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 1 week. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 2 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 3 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 4 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 5 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 6 weeks. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 1 month. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 2 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 3 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 4 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 5 months. Provided herein is the method wherein the depot injection provides a therapeutically effective concentration for a period of about 6 months or greater.

Provided herein is a method of treating opioid dependence in a patient in need thereof comprising administering a pharmaceutical composition comprising a compound disclosed in Table 3, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

TABLE 3

R X Chemical Name

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl palmitate

O (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (9Z,12Z)- octadeca-9,12-dienoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl )-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl palmitoleate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl myristoleate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl (Z)- hexadec-6-enoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl decanoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl undecanoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl dodecanoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl tridecanoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl tetradecanoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl pentadecanoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo- 2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2- e]isoquinolin-9-yl stearate

Other embodiments and uses will be apparent to one skilled in the art in light of the present disclosures. The following examples are provided merely as illustrative of various embodiments and shall not be construed to limit the invention in any way.

EXAMPLES

I. Chemical Synthesis

Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. Anhydrous solvents and oven-dried glassware were used for synthetic transformations sensitive to moisture and/or oxygen. Yields were not optimized. Reaction times are approximate and were not optimized. Column chromatography and thin layer chromatography (TLC) were performed on silica gel unless otherwise noted. Spectra are given in ppm (δ) and coupling constants, J are reported in Hertz. For proton spectra the solvent peak was used as the reference peak.

In some embodiments, opioid receptor antagonists prodrug compounds disclosed herein are synthesized according to the following examples.

General Scheme 1 for the Synthesis of Nalmefene Prodrugs.

General Scheme 2 for the Synthesis of Naltrexone Prodrugs.

Example 1: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl ((E)-octadec-9-en-1-yl) carbonate

To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (8 g, 21.28 mmol, 1 eq, HCl) in H₂O (100 mL) was added K₂CO₃ (8.82 g, 63.85 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. To a mixture of tetrabutylammonium sulfate (24.73 g, 21.28 mmol, 24.49 mL, 50% solution, 1 eq) in DCM (100 mL) then the later mixture was added to the former mixture. Iodomethyl (E)-octadec-9-en-1-yl carbonate (14.44 g, 31.92 mmol, 1.5 eq), obtained according to procedure described in Example 42B, was added and the mixture was stirred for 12 hours. The residue was concentrated in vacuum to remove the DCM then was dissolved by saturated solution of NaHCO₃ (100 mL). The aqueous phase was extracted with ethyl acetate 600 mL (200 mL*3). The combined organic phase was dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=20/1 to 1/1). The residue was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate 900 mL (300 mL*3). The combined organic phase was washed with brine (200 mL), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl [(E)-octadec-9-enyl] carbonate (5 g, 7.46 mmol, 35.03% yield) was obtained as a yellow oil. M+H⁺=665.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 1.

Example 2: Step 2A: Synthesis of (4-nitrophenyl) undecyl Carbonate

To a mixture of undecan-1-ol (40 g, 232.14 mmol, 1 eq) in DCM (600 mL) was added TEA (46.98 g, 464.29 mmol, 64.62 mL, 2 eq) (4-nitrophenyl) carbonochloridate (70.19 g, 348.22 mmol, 1.5 eq) was added to the former mixture portionwise under N₂. The mixture was stirred at 25° C. for 12 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography. Compound (4-nitrophenyl) undecyl carbonate (33.95 g, 100.62 mmol, 43.34% yield) was obtained as a yellow solid.

Step 2B: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Undecyl Carbonate

To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (15 g, 39.70 mmol, 1 eq, HCl) in DCM (150 mL) was added TEA (12.05 g, 119.09 mmol, 16.58 mL, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min, To a mixture of (4-nitrophenyl) undecyl carbonate (26.79 g, 79.39 mmol, 2 eq) in DCM (150 mL), then add to the former mixture, the mixture was stirred at 25° C. for 12 h. The residue was concentrated in vacuum to remove the DCM then was dissolved by saturated solution of NaHCO₃. The aqueous phase was extracted with ethyl acetate (200 mL*3). The combined organic phase was dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=40:1 to 1:1). The residue was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate (200 mL*3). The combined organic phase was washed with brine (500 mL), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. Compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] undecyl carbonate (7.91 g, 14.63 mmol, 36.85% yield) was obtained as a yellow oil. M+H⁺=540.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 2.

Example 3: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Undecyl Carbonate

To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (15 g, 39.91 mmol, 1 eq, HCl) in DCM (150 mL) was added TEA (12.11 g, 119.72 mmol, 16.66 mL, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min, To a mixture of (4-nitrophenyl) undecyl carbonate (26.93 g, 79.81 mmol, 2 eq) in DCM (150 mL), then add to the former mixture, the mixture was stirred at 25° C. for 12 h. The mixture was diluted with H₂O (800 mL), extracted with DCM (300 mL*3). The organic phase was washed with brine (300 mL), dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=40/1 to 1/1). The residue was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate (200 mL*3). The combined organic phase was washed with brine (500 mL), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] undecyl carbonate (11.40 g, 21.14 mmol, 52.97% yield) was obtained as a yellow oil. M+H⁺=538.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 3.

Example 4: Step 4A: Synthesis of Chloromethyl Undecyl Carbonate

To a mixture of undecan-1-ol (80 g, 464.29 mmol, 1 eq) and pyridine (73.45 g, 928.58 mmol, 74.95 mL, 2 eq) in DCM (600 mL) was added chloromethyl carbonochloridate (119.73 g, 928.58 mmol, 82.57 mL, 2 eq) dropwise at 0° C. under N₂. The mixture was stirred at 25° C. for 12h. The reaction mixture was extracted by DCM 1500 mL (500 mL*3). The organic phase was separated, washed with brine 30 mL (150 mL*2), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=50/1 to 1:1). Compound chloromethyl undecyl carbonate (80 g, 302.13 mmol, 65.07% yield) was obtained as a yellow oil.

Step 4B: Synthesis of Iodomethyl Undecyl Carbonate

To a mixture of chloromethyl undecyl carbonate (30 g, 113.30 mmol, 1 eq) in acetone (400 mL) was added NaHCO₃ (11.42 g, 135.96 mmol, 5.29 mL, 1.2 eq) and NaI (20.38 g, 135.96 mmol, 1.2 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12h in dark. The reaction mixture was partitioned between EtOAc (400 mL) and H₂O (400 mL). The organic phase was separated, washed with brine (80 mL), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0). Compound iodomethyl undecyl carbonate (60 g, 74.33% yield) was obtained as a yellow oil.

Step 4C: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Undecyl Carbonate

To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (8 g, 21.17 mmol, 1 eq, HCl) in H₂O (40 mL) was added K₂CO₃ (8.78 g, 63.52 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. Then was added tetrabutylammonium sulfate (24.60 g, 21.17 mmol, 24.36 mL, 50% solution, 1 eq) in DCM (40 mL) in one portion at 25° C. Then the mixture was added iodomethyl undecyl carbonate (15.08 g, 42.34 mmol, 2 eq) the mixture was stirred at 25° C. for 11.5h. The reaction mixture was partitioned between DCM 200 mL (100 mL*2) and H₂O 100 mL. The organic phase was separated, washed with brine 40 mL, dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=50/1 to 1:1). Then was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate (400 mL*3). washed with brine 300 mL, dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. Compound [(3R,4aS,7aR,12b5)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a, 13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl undecyl carbonate (6.9 g) was obtained as a yellow oil. M+H⁺=570.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 4.

Example 5: Synthesis of Example 5: (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Undecyl Carbonate

To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (8 g, 21.28 mmol, 1 eq, HCl) in H₂O (40 mL) was added K₂CO₃ (8.82 g, 63.85 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. Then was added tetrabutylammonium sulfate (24.73 g, 21.28 mmol, 24.49 mL, 50% solution, 1 eq) in DCM (40 mL) in one portion at 25° C. Then the mixture was added iodomethyl undecyl carbonate (15.16 g, 42.57 mmol, 2 eq), the mixture was stirred at 25° C. for 11.5h. The reaction mixture was partitioned between DCM 200 mL (100 mL*2) and H₂O 100 mL. The organic phase was separated, washed with brine 50 mL, dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=50/1 to 1:1). Then was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate (400 mL*3), washed with brine 300 mL, dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. Compound [(3R,4aS,7aS,12b)-3-(cyclopropylmethylm)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl undecyl carbonate (5.9 g) was obtained as a yellow oil. M+H⁺=568.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 5.

Example 6: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Dodecyl Carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 6.. Briefly, to a mixture of (3R,4aS,7aS,12b5)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2.5 g, 6.65 mmol, 1 eq, HCl) in DCM (10 mL) was added TEA (2.02 g, 19.95 mmol, 2.78 mL, 3 eq) and dodecyl carbonochloridate (2.48 g, 9.98 mmol, 1.5 eq). The mixture was stirred at −10° C. for 1 hour and then warmed to 25° C. for 4 hours under N₂. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, petroleum ether/ethyl acetate=5/1 to 1:1. The compound [(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate] was 98.570% pure and obtained as a yellow oil (1.5 g, 40.56% yield).

Example 7: Step 7A: Synthesis of chloromethyl (E)-octadec-9-en-1-yl Carbonate

To a mixture of (E)-octadec-9-en-1-ol (22 g, 81.94 mmol, 1 eq) and chloromethyl carbonochloridate (21.13 g, 163.89 mmol, 14.57 mL, 2 eq) in DCM (200 mL) was added pyridine (16.20 g, 204.86 mmol, 16.54 mL, 2.5 eq) dropwise at 0° C. under N₂. The reaction was stirred at 25° C. for 12 hr under N₂. The reaction mixture was quenched by addition H₂O 400 mL, and extracted with DCM 400 mL*1. The combined organic layers were washed with brine 300 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a oil. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0) to give product. Compound chloromethyl (E)-octadec-9-en-1-yl carbonate (50 g, 138.52 mmol, 84.52% yield) was obtained as a colorless oil.

Step 7B: Synthesis of iodomethyl (E)-octadec-9-en-1-yl carbonate

To a mixture of chloromethyl (E)-octadec-9-en-1-yl carbonate (30 g, 83.11 mmol, 1 eq) and NaI (18.69 g, 124.67 mmol, 1.5 eq) in acetone (300 mL) was added NaHCO₃ (8.38 g, 99.73 mmol, 3.88 mL, 1.2 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H₂O 500 mL and extracted with EtOAc 800 mL (400 mL*2). The combined organic layers were washed with NaCl aq. 400 mL, dried over, filtered and concentrated under reduced pressure to give target product. Compound iodomethyl (E)-octadec-9-en-1-yl carbonate (29 g, 64.10 mmol, 77.13% yield) was obtained as light yellow oil and was used into the next step without further purification.

Step 7C: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl ((E)-octadec-9-en-1-yl) carbonate

To a mixture of (4R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one; hydrochloride (8 g, 21.17 mmol, 1 eq) and K₂CO₃ (8.78 g, 63.52 mmol, 3 eq) in H₂O (200 mL) was stirred at 25° C. for 0.5 hr. Tetrabutylammonium sulfate (12.30 g, 21.17 mmol, 12.18 mL, 1 eq) in DCM (200 mL) was added the mixture and stirred for 0.5 hr at 25° C. Iodomethyl (E)-octadec-9-en-1-yl carbonate (14.37 g, 31.76 mmol, 1.5 eq) was added to the mixture and stirred for 11 hours. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H₂O (300 mL) and extracted with EtOAc (300 mL*3). The combined organic layers were washed with NaCl aq. (300 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=10/1 to 3:1) to give target product. Compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl [(E)-octadec-9-enyl] carbonate (8.08 g, 12.09 mmol, 57.12% yield,) was obtained as a colorless oil. M+H⁺=666.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 7.

Example 8: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (E)-octadec-9-enoate

To a mixture of (E)-octadec-9-enoic acid (6.28 g, 22.23 mmol, 1.2 eq) in DCM (100 mL) was added DMF (264.03 mg, 3.61 mmol, 277.93 uL, 0.195 eq) and oxalyl dichloride (8.46 g, 66.69 mmol, 5.84 mL, 3.6 eq) portionwise at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min, then concentrated under reduced pressure. DCM (100 mL) was added in the residue. To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (7 g, 18.53 mmol, 1 eq, HCl) in DCM (100 mL) was added TEA (3.75 g, 37.05 mmol, 5.16 mL, 2 eq), then the former mixture was added in the later mixture portionwise at 25° C. under N₂. The mixture was stirred at 25° C. for 12 hr. The residue was concentrated in vacuum to remove the DCM then was dissolved by saturated solution of NaHCO₃ (200 mL), The aqueous phase was extracted with ethyl acetate (100 mL*2). The combined organic phase was dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=40/1 to 1/1). The compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a, 13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] (E)-octadec-9-enoate (5.36 g, 8.11 mmol, 43.79% yield) was obtained as a yellow oil. M+H⁺=606.2 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 8.

Example 9: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl (E)-octadec-9-enoate

To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (10 g, 26.60 mmol, 1 eq, HCl) in H₂O (100 mL) was added K₂CO₃ (11.03 g, 79.81 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. To a mixture of tetrabutylammonium sulfate (30.91 g, 26.60 mmol, 30.61 mL, 50% solution, 1 eq) in DCM (100 mL) then the later mixture was added to the former mixture. Iodomethyl (E)-octadec-9-enoate (16.86 g, 39.91 mmol, 1.5 eq), obtained according to procedure described in Example 41B, was added and the mixture was stirred for 12 hours. The mixture was diluted with H₂O (100 mL), collect the organic phase, then the aqueous phase was extracted with Ethyl Acetate (300 mL*3), the organic phase was washed with brine (300 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=20/1 to 1/1). Then was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate (400 mL*3). The combined organic phase was washed with brine (500 mL), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl (E)-octadec-9-enoate (7.16 g, 11.15 mmol, 41.91% yield) was obtained as a yellow oil. M+H⁺=634.4 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 9.

Example 10: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexone prodrugs. 1.31 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 10. Briefly, to a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a, 13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (1.9 g, 5.57 mmol, 1 eq) in DCM (15 mL), cooled to −10° C. was added TEA (1.69 g, 16.70 mmol, 2.32 mL, 2 eq) and decyl carbonochloridate (2.46 g, 11.13 mmol, 2 eq). The mixture was stirred at 25° C. for 5 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography (SiO₂, petroleum ether/ethyl acetate=7/3 to 0:1) The compound [(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate] was 97.43% pure and obtained as a yellow oil with a 43.63% yield.

Example 11: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl Carbonate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexone prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 11. Briefly, to a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (2.5 g, 6.62 mmol, 1 eq, HCl) and TEA (1.34 g, 13.23 mmol, 1.84 mL, 2 eq) in DCM (15 mL) was added dodecyl carbonochloridate (1.56 g, 6.29 mmol, 0.95 eq). The mixture was stirred at −10° C. for 1 hr, then warmed to 25° C. and stirred for 4 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The residue product was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=7/3 to 0:1). The crude product was purified by prep-HPLC (column: Gemini 200*30 10μ; mobile phase—[water (10 mM NH₄HCO₃)—CAN]; B %70-100%, 12 minutes) The compound [(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate] was 99.497% pure and obtained as a white solid (1.5 g, 40.74% yield).

Example 12: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl stearate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.2 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 12.

Example 13: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (Z)-docos-13-enoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.3 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 13.

Example 14: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl docosanoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 14. Briefly, to a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2 g, 5.32 mmol, 1 eq, HCl) in DCM (10 mL) was added TEA (1.62 g, 15.96 mmol, 2.22 mL, 3 eq) and docosanoyl chloride (3.82 g, 10.64 mmol, 2 eq) one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=5/1 to 1:1) The residue was purified using prep-HPLC (TFA condition: column: Phenomenx luna (2) C18 250*50 10u; mobile phase: [water (0.1% TFA)-CAN]; B %: 65-95%, 20 minutes]). The compound [(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl docosanoate]was 97.01% pure and obtained as a white solid (1.5 g, 41.31% yield).

Example 15: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (E)-octadec-9-enoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.8 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 15. Briefly, to a mixture of (3R,4aS,7aS,12b5)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2 g, 5.32 mmol, 1 eq, HCl) in DCM (30 mL) was added TEA (1.08 g, 10.64 mmol, 1.48 mL, 2 eq) and (E)-octadec-9-enoyl chloride (1.92, 6.38 mmol, 1.2 eq). The mixture was stirred at 15° C. for 12 hr. The reaction mixture was mixed with H₂O (80 mL) and extracted with DCM (80 mL×3). The combined organic phase was washed with saturated NaHCO₃ solution (60 mL×2) and brine (60 mL×3), dried with anhydrous Na₂SO₄, filtered, and concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=10/1 to 1:1). The compound was purified again using a pre-HPLC column Phenomenex luna C18, 250×50 mm×10 μm; mobile phase: [water (0.1% TFA)-CAN]; B: 60-90%, 20 minutes). After pre-HPLC, the mixture was concentrated under reduced pressure. The aqueous phase was combined with NaHCO₃ to adjust the pH to 8, then the aqueous phase was extracted with ethyl acetate (30 mL×4). The combined organic phase was washed with brine (20 mL×1), dried with anhydrous Na₂SO₄, filtered and concentrated in a vacuum The compound [(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (E)-octadec-9-enoate] was 95% pure and obtained as a yellow oil (1.8 g, 29.57% yield).

Example 16: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl icosanoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.1 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 16. Briefly, to a solution of icosanoic acid (5 g, 16.00 mmol, 5.92 mL, 1 eq) in DCM (50 mL) was added DMF (116.93 mg, 1.6 mmol, 123.09 μL, 0.1 eq), cooled to 0° C., was add (COCl)₂ (2.34 g, 18.40 mmol, 1.61 mL, 1.15 eq). TEA (4.86 g, 48.80 mmol, 6.68 mL, 3 eq) and (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3.01 g, 8.00 mmol, 0.5 eq, HCl). The mixture was stirred at 25° C. for 12 hours. The reaction mixture was extracted with H2O (80 mL×1) and DCM (80 mL×2). The combined organic phase was washed with brine (60 mL×3), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The compound was purified by column chormoatrography (SiO₂, petroleum ether/ethyl acetate=10/1 to 1:1. The compound [4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl icosanoate] was 100% pure and obtained as a white solid (1.1 g, 10.84% yield).

Example 17: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Octyl Carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs and was obtained as an oil. 1.5 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 17.

Example 18: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs and was obtained as an oil. 1.5 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 18.

Example 19: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Hexadecyl Carbonate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.8 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 19. Briefly, to a solution of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a, 13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (5 g, 13.30 mmol, 1 eq) in DCM (50 mL), cooled to −10° C., TEA (4.04 g, 39.91 mmol, 5.55 mL, 3 eq) and hexadecyl carbonochloridate (8.11 g, 26.60 mmol, 2 eq) was added. Then, the mixture was stirred at 25° C. for 5 hours under N₂ atmosphere. The reaction mixture was extracted with H₂O (80 mL×l) and DCM (80 mL×2). The combined organice phase was washed with brine (60 mL×3), dried with anhydrous Na₂SO₄, filtered and concentration in vacuum. The reside and compound was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=10/1 to 1:1) The residue was purified by prep-HPLC (TFA condition: column—Phenomenex lune C18 250×50 mm×10 μm; mobile phase—[water (0.1% TFA)-CAN]; B % 65-95%, 20 minutes). NaHCO₃ was added to adjust pH to 8, and then extracted with EtOAc (20 mL×3). The organic layer was evaporated under reduced pressure to get the final product. The compound [4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate] was 99.723% pure and was obtained as a white solid (1.8 g, 12.33% yield).

Example 20: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.4 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 20.

Example 21: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexone prodrugs. 2.15 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 21. Briefly, to a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (2 g, 5.29 mmol, 1 eq, HCl0 in DCM (20 mL), cooled to −10° C., was added TEA (1.61 g, 15.88 mmol, 2.21 mL, 3 eq) and hexadecyl carbonochloridate (3.23 g, 10.59 mmol, 2 eq). Then, the mixture was stirred at 25° C. for 5 hours under N₂ atmosphere. The reaction mixture was extracted with H2O (80 mL×1) and DCM (80 mL×2). The residue was purified by prep-HPLC (TFA condition: column—Phenomenex luna (2) C18 250×50 mm×10 μm; mobile phase—[water (0.1% TFA)-CAN]; B % 60-90%, 20 minutes). NaHCO₃ was added to adjust pH to 8, and then extracted with EtOAc (20 mL×3). The organic layer was evaporated under reduced pressure to get the final product. The compound [(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate] was 97.669% pure and obtained as a white solid (2.15 g, 65.06% yield)

Example 22: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (Z)-docos-13-enoate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexone prodrugs. 2.17 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 22.

Example 23: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Octyl Carbonate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexone prodrugs. 1.33 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 23.

Example 24: Step 24A: Synthesis of chloromethyl dodecyl carbonate

To a mixture of dodecan-1-ol (30 g, 161.00 mmol, 1 eq) in DCM (300 mL) was added TEA (32.58 g, 322.00 mmol, 44.82 mL, 2 eq) and chloromethyl carbonochloridate (41.52 g, 322.00 mmol, 28.63 mL, 2 eq) in one portion at 0° C. under N₂. The mixture was heated to 25° C. and stirred for 12 hr. The reaction mixture was quenched by addition water 200 mL at 25° C., and then extracted with DCM 100 mL (50 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethyl dodecyl carbonate (10.3 g, 36.94 mmol, 22.95% yield) was obtained as a colorless oil.

Step 24B: Synthesis of iodomethyl dodecyl carbonate

To a mixture of chloromethyl dodecyl carbonate (10 g, 35.87 mmol, 1 eq) in acetone (100 mL) was added NaHCO₃ (3.62 g, 43.04 mmol, 1.67 mL, 1.2 eq) and NaI (6.45 g, 43.04 mmol, 1.2 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12 hours in dark. The reaction mixture was filtered to remove the insoluble and concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate 50 mL and the organic layer was washed with water 60 mL (30 mL*2), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound dodecyl iodomethyl carbonate (12.6 g, crude) was obtained as a light red oil. The crude product dodecyl iodomethyl carbonate was used into the next step without further purification.

Step 24C: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl dodecyl carbonate

To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (4 g, 10.64 mmol, 1 eq, HCl) in H₂O (20 mL) was added K₂CO₃ (4.41 g, 31.92 mmol, 3 eq) and the mixture was stirred for 30 min at 20° C. Tetrabutylammonium sulfate (12.37 g, 10.64 mmol, 12.24 mL, 1 eq) and DCM (20 mL) were added to the mixture and the mixture was stirred for 10 min at 20° C. Dodecyl iodomethyl carbonate (9.46 g, 25.54 mmol, 2.4 eq) was added to the mixture in one portion at 20° C. under N₂. The mixture was stirred at 20° C. for 12 hours. The reaction mixture was diluted with water 20 mL and extracted with DCM 20 mL (10 mL*2). The combined organic layers were dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 10:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[32-e]isoquinoline-9-yl]oxymethyl dodecyl carbonate (2.92 g, 5.00 mmol, 47.02% yield) was obtained as a colorless oil. M+H+=582.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 24.

Example 25: Step 25A: Synthesis of Chloromethyl Tetradecyl Carbonate

To a mixture of tetradecan-1-ol (30 g, 139.93 mmol, 1 eq) in DCM (300 mL) was added TEA (28.32 g, 279.87 mmol, 38.95 mL, 2 eq) and chloromethyl carbonochloridate (36.09 g, 279.87 mmol, 24.89 mL, 2 eq) in one portion at 0° C. under N₂, then heated to 25° C. for 12 hr. The reaction mixture was quenched by addition water 200 mL at 25° C., and then extracted with DCM 100 mL (50 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethyl tetradecyl carbonate (11 g, 35.85 mmol, 25.62% yield) was obtained as a colorless oil.

Example 25B: Synthesis of Iodomethyl Tetradecyl Carbonate

To a mixture of chloromethyl tetradecyl carbonate (11.1 g, 36.17 mmol, 1 eq) in acetone (100 mL) was added NaHCO₃ (3.04 g, 36.17 mmol, 1.41 mL, 1 eq) and NaI (5.42 g, 36.17 mmol, 1 eq) in one portion at 15° C. under N₂. The mixture was stirred at 15° C. for 12 h. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with ethyl acetate 40 mL and washed with water 40 mL (20 mL*2). The organic layers were dried, filtered and concentrated under reduced pressure to give a residue. The crude product iodomethyl tetradecyl carbonate (13.1 g, 32.89 mmol, 90.92% yield) was obtained as light red oil and used into the next step without further purification.

Step 25C: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Tetradecyl Carbonate

To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3 g, 7.98 mmol, 1 eq, HCl) and iodomethyl tetradecyl carbonate (7.63 g, 19.15 mmol, 2.4 eq) in H₂O (30 mL) was added K₂CO₃ (3.31 g, 23.94 mmol, 3 eq) and the mixture was stirred for 0.5 h at 15° C. After 0.5 h, tetrabutylammonium sulfate (4.64 g, 7.98 mmol, 4.59 mL, 1 eq) and DCM (30 mL) were added to the mixture and the mixture was stirred for 10 min at 15° C. After 10 min, iodomethyl tetradecyl carbonate (7.63 g, 19.15 mmol, 2.4 eq) was added to the mixture in one portion at 15° C. under N₂. The mixture was stirred at 15° C. for 12 h. The residue was diluted with water 10 mL and extracted with DCM 20 mL (10 mL*2). The combined organic layers were dried, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 10:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl tetradecyl carbonate (2.0 g, 3.25 mmol, 40.76% yield) was obtained as a colorless oil. M+H⁺=610.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 25.

Example 26: Step 26A: Synthesis of Chloromethyl (E)-octadec-9-enoate

To a mixture of (E)-octadec-9-enoic acid (2 g, 7.08 mmol, 1 eq) in DCM (15 mL) and H₂O (8 mL) was added NaHCO₃ (2.38 g, 28.32 mmol, 1.10 mL, 4 eq) and tetrabutylammonium sulfate (822.29 mg, 708.06 umol, 50% solution, 0.1 eq) in one portion at 25° C. under N₂, then the mixture was cooled to 0° C. Chloro(chlorosulfonyloxy)methane (1.17 g, 7.08 mmol, 1 eq) in DCM (10 mL) was added to the mixture at 0° C. The mixture was heated to 25° C. and stirred for 18 hours. The reaction mixture was extracted with DCM 30 mL (15 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound chloromethyl (E)-octadec-9-enoate (1.97 g, 5.95 mmol, 84.07% yield) was obtained as a white solid and was used into the next step without purification.

Step 26B: Synthesis of Iodomethyl (E)-octadec-9-enoate

To a mixture of chloromethyl (E)-octadec-9-enoate (14.5 g, 43.82 mmol, 1 eq) in acetone (140 mL) was added NaHCO₃ (4.42 g, 52.58 mmol, 2.04 mL, 1.2 eq) and NaI (7.88 g, 52.58 mmol, 1.2 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12 hours in dark. The reaction mixture was filtered to remove the insoluble and concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate (100 mL) and the organic layer was washed with brine 100 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound iodomethyl (E)-octadec-9-enoate (18.6 g, crude) was obtained as a brown oil and was used into the next step without purification.

Step 26C: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl (E)-octadec-9-enoate

To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (10.09 g, 26.71 mmol, 1 eq, HCl) in H₂O (100 mL) was added K₂CO₃ (11.07 g, 80.12 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. A mixture of tetrabutylammonium sulfate (15.01 g, 12.92 mmol, 50% solution, 4.84e-1 eq) in DCM (100 mL) then the later mixture was added to the former mixture. Iodometyl (E)-octadec-9-enoate (16.92 g, 40.06 mmol, 1.5 eq) was added and the mixture was stirred for 12 hours. The mixture was diluted with H₂O (800 mL), collect the organic layer, then was extracted with Ethyl Acetate (300 mL*3). All the organic phase was washed with brine (300 mL), dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=20/1 to 1/1). The residue was further purified by prep-HPLC, MeOH as solvent, select conventional reverse phase separation as method, separation system is TFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase was extracted with ethyl acetate (400 mL*3). The combined organic phase was washed with brine (500 mL), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl (E)-octadec-9-enoate (10.20 g, 15.85 mmol, 59.35% yield) was obtained as a yellow oil. M+H⁺=636.4 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 26.

Example 27: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Tetradecyl Carbonate

To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (4.5 g, 11.91 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (4.94 g, 35.73 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. tetrabutylammonium sulfate (13.84 g, 11.91 mmol, 13.70 mL, 50% solution, 1 eq) and DCM (30 mL) were added to the mixture in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 10 min. Iodomethyl tetradecyl carbonate (11.38 g, 28.58 mmol, 2.4 eq) was added to the mixture in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12 hours. The reaction mixture was extracted with DCM 30 mL (15 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 5:1). Compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl tetradecyl carbonate (2.8 g, 4.53 mmol, 38.05% yield, 99% purity) was obtained as a colorless oil. ¹H NMR (400 MHz, CDCl₃): see FIG. 27.

Example 28: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Icosyl Carbonate

To a mixture of icosyl (4-nitrophenyl) carbonate (9.87 g, 21.28 mmol, 4 eq) in DCM (40 mL) was added TEA (538.40 mg, 5.32 mmol, 740.58 uL, 1 eq) and (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2 g, 5.32 mmol, 1 eq, HCl) in one portion at 15° C. under N₂. The mixture was stirred at 15° C. for 12 hr. The reaction mixture was extracted with H₂O mL (20 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC. Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] icosyl carbonate (1.6 g, 2.35 mmol, 44.25% yield) was obtained as a white solid. ¹H NMR (400 MHz, CDCl₃): see FIG. 28.

Example 29: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Dodecyl Carbonate

To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (4.5 g, 11.91 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (4.94 g, 35.73 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. tetrabutylammonium sulfate (13.84 g, 11.91 mmol, 13.70 mL, 50% solution, 1 eq) and DCM (30 mL) were added to the mixture in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 10 min. Dodecyl iodomethyl carbonate (10.58 g, 28.58 mmol, 2.4 eq) was added to the mixture in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 12 hours. The reaction mixture was extracted with DCM 30 mL (15 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 5:1). Compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl dodecyl carbonate (3.1 g, 5.26 mmol, 44.19% yield, 99.1% purity) was obtained as a colorless oil. ¹H NMR (400 MHz, CDCl₃): see FIG. 29.

Example 30: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Tridecyl Carbonate

To a mixture of (4-nitrophenyl) tridecyl carbonate (5.83 g, 15.96 mmol, 2 eq) in DCM (50 mL) was added TEA (2.42 g, 23.94 mmol, 3.33 mL, 3 eq) and (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3 g, 7.98 mmol, 1 eq, HCl) in one portion at 15° C. under N2. The mixture was stirred at 15° C. for 12 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1/0 to 3:1). [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] tridecyl carbonate (2.3 g, 4.07 mmol, 50.94% yield) was obtained as a colorless oil. ¹H NMR (400 MHz, CDCl₃): see FIG. 30.

Example 31: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Tetradecyl Carbonate

To a solution of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3 g, 7.98 mmol, 1 eq, HCl) in DCM (20 mL) was added TEA (1.62 g, 15.96 mmol, 2.22 mL, 2 eq) and tetradecyl carbonochloridate (2.21 g, 7.98 mmol, 1 eq). The mixture was stirred at 15° C. for 12 hr. The mixture was concentrated under reduced pressure. The residue was mixed with H₂O (80 mL) and extracted with DCM (80 mL*3). The combined organic phase was washed with saturated NaHCO₃ solution (60 mL*2) and brine (60 mL*3), dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=4/1 to 0:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] tetradecyl carbonate (2 g, 3.41 mmol, 42.79% yield) was obtained as a colorless oil. M+H⁺=580.4 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 31.

Example 32: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Pentadecyl Carbonate

To a mixture of (4-nitrophenyl) pentadecyl carbonate (6.28 g, 15.96 mmol, 2 eq) in DCM (30 mL) was added TEA (2.42 g, 23.94 mmol, 3.33 mL, 3 eq) and [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3 g, 7.98 mmol, 1 eq, HCl) in one portion at 15° C. under N₂. The mixture was stirred at 15° C. for 12 hr. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 2:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl] pentadecyl carbonate (2.6 g, 1.80 mmol, 22.49% yield) was obtained as a white solid. M+H⁺=594.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 32.

Example 33: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Octadecyl Carbonate

To a solution of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2 g, 5.32 mmol, 1 eq, HCl) in DCM (30 mL) was added TEA (1.62 g, 15.96 mmol, 2.22 mL, 3 eq) and (4-nitrophenyl) octadecyl carbonate (3.48 g, 7.98 mmol, 1.5 eq). The mixture was stirred at 15° C. for 12 hr. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=10/1 to 1:1) and then by pre-HPLC. Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]octadecyl carbonate (0.8 g, 1.22 mmol, 22.93% yield) was obtained as a yellow oil. M+H⁺=636.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 33.

Example 34: Step 34A: Synthesis of Chloromethyl Hexadecyl Carbonate

To a mixture of hexadecan-1-ol (30 g, 123.74 mmol, 1 eq) in DCM (200 mL) was added TEA (25.04 g, 247.48 mmol, 34.45 mL, 2 eq) and chloromethyl carbonochloridate (31.91 g, 247.48 mmol, 22.01 mL, 2 eq) in one portion at 0° C. under N₂. The mixture was heated to 20° C. and stirred for 12 hours. The reaction mixture was quenched by addition water 50 mL at 20° C., and then extracted with DCM 100 mL (50 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethyl hexadecyl carbonate (18 g, 53.74 mmol, 43.43% yield) was obtained as a white solid.

Step 34B: Synthesis of Iodomethyl Hexadecyl Carbonate

To a mixture of chloromethyl hexadecyl carbonate (8 g, 23.89 mmol, 1 eq) in acetone (50 mL) was added NaHCO₃ (2.41 g, 28.66 mmol, 1.11 mL, 1.2 eq) and NaI (4.30 g, 28.66 mmol, 1.2 eq) in one portion at 15° C. under N₂. The mixture was stirred at 15° C. for 12 h. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with ethyl acetate 20 mL and washed with water 20 mL (10 mL*2). The organic layers were dried, filtered and concentrated under reduced pressure to give a residue. The crude product hexadecyl iodomethyl carbonate (9 g, crude) was obtained as a light red solid and used into the next step without further purification. Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloxy)methyl hexadecyl carbonate

To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (2.75 g, 7.32 mmol, 1 eq, HCl) and hexadecyl iodomethyl carbonate (7.49 g, 17.56 mmol, 2.4 eq) in H₂O (25 mL) was added K₂CO₃ (3.03 g, 21.95 mmol, 3 eq) and stirred for 0.5 h at 15° C. After 30 min, tetrabutylammonium sulfate (4.25 g, 7.32 mmol, 4.21 mL, 1 eq) and DCM (25 mL) were added to the mixture and the mixture was stirred for more 10 min. After 10 min, hexadecyl iodomethyl carbonate (7.49 g, 17.56 mmol, 2.4 eq) was added to the mixture in one portion at 15° C. under N₂. The mixture was stirred at 15° C. for 12 hours. The residue was diluted with water 10 mL and extracted with DCM 20 mL (10 mL*2). The combined organic layers were dried, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 10:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl hexadecyl carbonate (2.0 g, 3.10 mmol, 42.38% yield) was obtained as a colorless oil. M+H⁺=638.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 34.

Example 35: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Decyl Carbonate

(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl decyl carbonate is prepared in a manner analogous to Example 5. To a mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol in H₂O is added K₂CO₃ in one portion at 25° C. under N₂. The mixture is stirred at 25° C. for 30 min. Then is added tetrabutylammonium sulfate in DCM in one portion at 25° C. Then is added to the reaction mixture iodomethyl decyl carbonate, the mixture is stirred at 25° C. until the reaction is complete. The reaction mixture is then subjected to workup and the desired product isolated by chromatography as in Example 5. ¹H NMR (400 MHz, CDCl₃): see FIG. 35.

Example 36: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Oleate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 36.

Example 37: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z)-octadeca-9,12-dienoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.3 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 37.

Example 38: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3,3-dimethylbutanoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.2 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 38.

Example 39: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3-cyclopentylpropanoate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs. 1.18 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 39.

Example 40: Synthesis of (4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl tert-butylcarbamate

The title compound was synthesized according to the general Scheme 1 for the synthesis of nalmefene prodrugs and was obtained as a solid. 3.0 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 40.

Example 41: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl Oleate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexone prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 41.

Example 42: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3,3-dimethylbutanoate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexome prodrugs. 1.3 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 42.

Example 43: Synthesis of (4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl 3-cyclopentylpropanoate

The title compound was synthesized according to the general Scheme 2 for the synthesis of naltrexone prodrugs. 1.3 g; ¹H NMR (400 MHz, CDCl₃): see FIG. 43.

Example 44: Step 44A: Synthesis of Chloromethyl Dodecanoate

To a mixture of dodecanoic acid (20 g, 99.84 mmol, 1 eq) in DCM (60 mL) and H₂O (80 mL) was added NaHCO₃ (33.55 g, 399.37 mmol, 15.53 mL, 4 eq) and tetrabutylammonium sulfate (11.60 g, 9.98 mmol, 11.49 mL, 50% purity, 0.1 eq) in one portion at 25° C. under N2, then the mixture was cooled to 0° C. The reactant of chloro(chlorosulfonyloxy)methane (16.47 g, 99.84 mmol, 1 eq) in DCM (20 mL) were added to the mixture in one portion at 0° C. The mixture was heated to 25° C. and stirred for 18 hours. The reaction mixture was extracted with DCM 50 mL (25 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethyl dodecanoate (10.8 g, 43.41 mmol, 43.48% yield) was obtained as a colorless oil.

Step 44B: Synthesis of Iodomethyl Dodecanoate

A mixture of chloromethyl dodecanoate (9 g, 36.18 mmol, 1 eq) in acetone (80 mL) was degassed and purged with N₂ for 3 times, and then NaHCO₃ (3.04 g, 36.18 mmol, 1.41 mL, 1 eq) and NaI (5.42 g, 36.18 mmol, 1 eq) was added to the mixture in dark, and the result mixture was stirred at 15° C. for 12 h under N₂ atmosphere in dark. The reaction mixture was filtered and concentrated under reduced pressure to remove solvent. The residue was diluted with H₂O O 50 mL and extracted with EtOAc 120 mL. The combined organic layers were washed with H₂O 100 mL (50 mL*2), dried, filtered and concentrated under reduced pressure to give a residue. Compound iodomethyl dodecanoate (9 g, crude) was obtained as a yellow liquid and used into the next step without further purification.

Step 44C: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Dodecanoate

A mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3.5 g, 10.31 mmol, 1 eq), K₂CO₃ (4.28 g, 30.93 mmol, 3 eq) in H₂O (40 mL) was stirred at 15° C. for 30 min and then tetrabutylammonium sulfate (5.99 g, 10.31 mmol, 5.93 mL, 1 eq) and DCM (20 mL) was added to the mixture and a solution of iodomethyl dodecanoate (8.42 g, 24.75 mmol, 2.4 eq) in DCM (20 mL) was added to the mixture and degassed and purged with N₂ for 3 times, and then the mixture was stirred at 15° C. for 11.5 h under N₂ atmosphere. The reaction mixture was diluted with H₂O 20 mL and extracted with DCM 20 mL. The combined organic layers were dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 20:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl dodecanoate (1.51 g, 2.70 mmol, 26.14% yield) was obtained as a colorless oil. M+H+=552.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 44.

Example 45: Step 45A Synthesis of Chloromethyl Tetradecanoate

To a mixture of tetradecanoic acid (20 g, 87.58 mmol, 1 eq) in H₂O (80 mL) was added NaHCO₃ (29.43 g, 350.31 mmol, 13.62 mL, 4 eq) and tetrabutylammonium sulfate (10.18 g, 8.76 mmol, 10.08 mL, 50% solution, 0.1 eq) and DCM (60 mL) under N₂. The mixture was cooled to 0° C. The reactant chloro(chlorosulfonyloxy)methane (14.45 g, 87.58 mmol, 1 eq) in DCM (20 mL) was added to the mixture in one portion at 0° C. under N₂. The mixture was heated to 25° C. and stirred for 18 hours. The reaction mixture was extracted with DCM 50 mL (25 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethyl tetradecanoate (15.5 g, 55.99 mmol, 63.93% yield) was obtained as a colorless oil.

Step 45B: Synthesis of Iodomethyl Tetradecanoate

A mixture of chloromethyl tetradecanoate (8 g, 28.90 mmol, 1 eq) in acetone (70 mL) was degassed and purged with N₂ for 3 times, and then NaHCO₃ (2.43 g, 28.90 mmol, 1.12 mL, 1 eq) and NaI (4.33 g, 28.90 mmol, 1 eq) was added to the mixture in dark, the result mixture was stirred at 15° C. for 12 hr under N₂ atmosphere in dark. The reaction mixture was filtered and concentrated under reduced pressure to remove solvent. The residue was diluted with H₂O 50 mL and extracted with EtOAc 120 mL. The combined organic layers were washed with H₂O 100 mL (50 mL*2), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. Compound iodomethyl tetradecanoate (9 g, crude) was obtained as a yellow solid.

Step 45C: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Tetradecanoate

A mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3.5 g, 9.31 mmol, 1 eq, HCl), K₂CO₃ (3.86 g, 27.93 mmol, 3 eq) in H₂O (30 mL) was stirred at 15° C. for 30 min, and then tetrabutylammonium sulfate (5.41 g, 9.31 mmol, 5.36 mL, 1 eq) and DCM (15 mL) was added to the mixture and a solution of iodomethyl tetradecanoate (8.23 g, 22.35 mmol, 2.4 eq) in DCM (15 mL) was added to the mixture and degassed and purged with N₂ for 3 times, and then the mixture was stirred at 15° C. for 11.5 h under N₂ atmosphere. The reaction mixture was diluted with H₂O 20 mL and extracted with DCM 20 mL. The combined organic layers were dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 20:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl tetradecanoate (1.5 g, 2.54 mmol, 27.26% yield) was obtained as a colorless oil. M+H⁺=580.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 45.

Example 46: Step 46A: Synthesis of Chloromethyl Hexadecanoate

To a mixture of palmitic acid (20 g, 78.00 mmol, 23.47 mL, 1 eq) in DCM (60 mL) and H₂O (80 mL) was added NaHCO₃ (26.21 g, 311.98 mmol, 12.13 mL, 4 eq) and tetrabutylammonium sulfate (9.06 g, 7.80 mmol, 8.97 mL, 50% purity, 0.1 eq) in one portion at 25° C. under N₂ and then the mixture was cooled to 0° C. The reactant of chloro(chlorosulfonyloxy)methane (12.87 g, 78.00 mmol, 1 eq) in DCM (20 mL) were added to the mixture in one portion at 0° C. The mixture was heated to 25° C. and stirred for 18 hours. The reaction mixture was extracted with DCM 50 mL (25 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethyl hexadecanoate (17.6 g, 57.72 mmol, 74.01% yield) was obtained as a white solid.

Step 46B: Synthesis of iodomethyl hexadecanoate

A mixture of chloromethyl hexadecanoate (3 g, 9.84 mmol, 1 eq) in acetone (30 mL) was degassed and purged with N₂ for 3 times at 15° C. in dark, and then the mixture was added NaHCO₃ (826.58 mg, 9.84 mmol, 382.68 uL, 1 eq) and NaI (1.47 g, 9.84 mmol, 1 eq) and stirred at 15° C. for 12 h under N₂ atmosphere in dark. The reaction mixture filtered and the filtrate was concentrated under reduced pressure to remove solvent. The residue was diluted with H₂O 20 mL and extracted with EtOAc 60 mL. The combined organic layers were washed with H₂O 40 mL (20 mL*2), dried, filtered and concentrated under reduced pressure to give a residue. Compound iodomethyl hexadecanoate (3.5 g, crude) was obtained as a yellow solid and used into the next step without further purification.

Step 46C: Synthesis of (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Hexadecanoate

A mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (1.8 g, 4.79 mmol, 1 eq, HCl), K2CO3 (1.99 g, 14.37 mmol, 3 eq) in H2O (15 mL) was stirred for 30 min, and tetrabutylammonium sulfate (2.78 g, 4.79 mmol, 2.75 mL, 1 eq) and DCM (7.5 mL) was added to the mixture, and a solution of iodomethyl hexadecanoate (4.56 g, 11.49 mmol, 2.4 eq) in DCM (7.5 mL) was added to the mixture and degassed and purged with N2 for 3 times, and then the mixture was stirred at 15° C. for 11.5 h under N2 atmosphere. The reaction mixture was diluted with H2O 10 mL and extracted with DCM 10 mL. The combined organic layers were dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:0 to 20:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl hexadecanoate (1.6 g, 24.35% yield) was obtained as a colorless oil. M+H+=608.6 (LCMS). 1H NMR (400 MHz, CDCl₃): see FIG. 46.

Example 47: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Hexadecyl Carbonate

To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (5.45 g, 14.42 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (5.98 g, 43.27 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. tetrabutylammonium sulfate (16.76 g, 14.42 mmol, 16.59 mL, 50% solution, 1 eq) and DCM (30 mL) were added to the mixture at 25° C. and the mixture was stirred for 10 min at 25° C. hexadecyl iodomethyl carbonate (14.76 g, 34.62 mmol, 2.4 eq) was added to the mixture in one portion at 25° C. and the mixture was stirred at 25° C. for 12 hr. The reaction mixture was extracted with DCM 30 mL (15 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under the reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 5:1). Compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl hexadecyl carbonate (4.68 g, 7.29 mmol, 50.56% yield) was obtained as a colorless oil. M+H+=640.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 47.

Example 48: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Dodecanoate

A mixture of (3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol (3.5 g, 10.31 mmol, 1 eq), K₂CO₃ (4.28 g, 30.93 mmol, 3 eq) in H₂O (40 mL) was stirred at 15° C. for 30 min and then tetrabutylammonium sulfate (5.99 g, 10.31 mmol, 5.93 mL, 1 eq) and DCM (20 mL) was added to the mixture and a solution of iodomethyl dodecanoate (8.42 g, 24.75 mmol, 2.4 eq) in DCM (20 mL) was added to the mixture and degassed and purged with N₂ for 3 times, and then the mixture was stirred at 15° C. for 11.5 h under N₂ atmosphere. The reaction mixture was diluted with H₂O 20 mL and extracted with DCM 20 mL. The combined organic layers were dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 20:1). Compound [(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl dodecanoate (1.51 g, 2.70 mmol, 26.14% yield) was obtained as a colorless oil. M+H+=554.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 48.

Example 49: Synthesis of (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl Hexadecanoate

To a mixture of (3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one (5 g, 13.23 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (5.49 g, 39.70 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 30 min. tetrabutylammonium sulfate (15.38 g, 13.23 mmol, 15.22 mL, 50% solution, 1 eq) and DCM (30 mL) were added to the mixture in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 10 min. Iodomethyl hexadecanoate (12.59 g, 31.76 mmol, 2.4 eq) was added to the mixture in one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12 hr. The reaction mixture was extracted with DCM 30 mL (15 mL*2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 5:1). Compound [(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a, 13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl hexadecanoate (5.2 g, 6.14 mmol, 46.40% yield) was obtained as a light yellow oil. M+H⁺=610.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 49.

Examples 50-61. Compounds 50-61 Listed in Table 2 can be Prepared According to General Scheme 1 for the Synthesis of Nalmefene Prodrugs or General Scheme 2 for the Synthesis of Naltrexone Prodrugs with Suitable Starting Materials

II. Biological Evaluation

Example 1: Plasma and Liver S9 Fraction Stability Assay

Plasma stability determination of the test compounds in rat, dog, cynomolgus monkey and human plasma is performed using HPLC-MS. For rat, incubations are carried out in 96-well polypropylene plates in 5 aliquots of 70 μL each (one for each time point). Test compounds (10 μM, final solvent concentration 1%) are incubated at 37° C. Five time points are analyzed (0, 15, 120, 480 and 1440 min). For dog, monkey and human, test compounds (2 μM, final solvent concentration 1%) were also incubated at 37° C. and analyzed at five time points (0, 10, 30, 60 and 120 min). All incubations are performed in duplicates. The samples are analyzed by HPLC-MS. The percentage of parent compound remaining after incubation in plasma is determined. Nalmefene dodecanoate and nalmefene palmitate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202). Results are provided in Table 4a-d.

TABLE 4a Rat Plasma Stability % remaining at time point in minutes 0 15 120 480 1440 Compound min min min min min 36 100 74.4 66.3 39.8 19.1 6 100 31.3 7.6 4 1.6 53 100 46.85 19.6 7.3 1.9 11 100 0.7 0.3 0.2 0 54 100 98.8 62.4 45.6 19.4 14 100 94 112 107 120 15 100 72 69.3 52 35 17 100 0 0 0 0 18 100 0.6 0.3 0.1 0 19 100 70.1 71.5 43.8 33.3 55 100 77.3 47.2 27.1 15.6 23 100 0 0 0 0 10 100 0 0 0 0 21 100 73.5 68.3 59.8 40.9 50 100 114.5 102.7 83.3 73.9 51 100 107.4 105.3 88.2 92.7 8 100 44.2 34.3 15.3 5.2 57 100 10.5 3.2 1.2 0 58 100 37 20 2.9 1.8 30 100 65.7 40.3 30.5 22.2 31 100 123.2 73.5 54.4 46.4 24 100 32 11.8 8.2 3 25 100 82.6 69.3 62.2 51.7 59 100 5.9 0.7 0.2 0 56 100 73.1 56.7 35.8 16.8 Aripiprazole 100 84 85.5 76.4 46 Lauroxil (plasma cleavage control) Enalapril maleate 100 44 2.9 8.2 4.8 salt (plasma cleavage control)

TABLE 4b Dog Plasma stability % remaining at time point in minutes Compound 0 min 15 min 120 min 480 min 1440 min 6 100 89.1 96.9 107.2 101.2 15 100 82.5 80.6 90.1 86.1 24 100 94.7 104.5 115.7 116.2 Aripiprazole lauroxil 100 103 81.3 78.6 74.3 Paliperidone palmitate 100 91.2 88.4 95.9 93.1

TABLE 4c Monkey Plasma stability % remaining at time point in minutes Compound 0 min 15 min 120 min 480 min 1440 min 6 100 88.7 74.3 68.4 70.1 15 100 74.5 72.7 80.6 76.5 24 100 90.7 93.5 96.2 97.8 Aripiprazole lauroxil 100 95.2 94.5 75.5 83.2 Paliperidone palmitate 100 92 89.5 98.4 92.5

TABLE 4d Human Plasma stability % remaining at time point in minutes Compound 0 min 15 min 120 min 480 min 1440 min 6 100 97.6 102 109.8 115.1 15 100 83.3 82.5 107.1 109.6 24 100 94 102.3 105.3 112.5 Aripiprazole lauroxil 100 99.7 80 73.2 75.5 Paliperidone palmitate 100 103.7 97.6 109.1 104.1

Liver S9 fraction stability determination of the test compounds in dog, cynomolgus monkey and human is performed using HPLC-MS. Test compound (2 μM, 0.1% DMSO, 1% Methanol final concentration) was assessed for stability in a 50 μl phosphate buffer containing 1.0 mg/ml S9 protein from each of the three species and 5 mM D-saccharic acid-1, 4-lactone. Samples were incubated at 37° C. for 60 minutes and the % compound remaining was assessed.

TABLE 4e Liver S9 Fraction Stability % remaining at 60 minutes Compound Dog Monkey Human 6 17.1 0 5.3 15 80.9 76.7 59.8 24 3.6 3 3.5 Aripiprazole lauroxil 66.1 56.6 47.8 Paliperidone palmitate 56.4 57.4 47.8

Example 2: Opioid Receptor Binding Assay

Receptor binding assays were performed to assess the ability of compounds to inhibit binding to radiolabeled ligand. First, the IC50 values were determined for select compounds for all 3 opioid receptor subtypes (DOR, MOR and KOR) and compared these values to that of the parent molecule, Nalmefene. The general observation is that prodrug derivatization greatly reduces the binding affinity to the opioid receptors, in some cases by several orders of magnitude.

Apparatus

Unifilter-96 GF/C filter plates, Perkin Elmer (Cat#6005174)

96 well conical polypropylene plates, Agilent (Cat#5042-385)

TopSeal-A sealing film, Perkin Elmer (Cat#6005250)

TopCount NXT HTS, (PerkinElmer)

MicroBeta² (PerkinElmer)

Cell harvest C961961, (Perkin Elmer)

Reagents

The stable cell lines were established and prepared cell membrane obtained using these cell lines.

³H-diprenophrine (PerkinElmer, Cat: NET1121250UC, Lot: 2143599)

³H-DAMGO (PerkinElmer, Cat: NET902250UC, Lot: 2139100)

³H-DADLE (PerkinElmer, Cat: NET648250UC, Lot: 2060549)

Tris base (Sigma, Cat: T6066-1KG), prepare 1M stock and adjust pH to 7.4.

0.5M EDTA (Invitrogen, Cat: 15575-038)

1M MgCl₂ (Sigma, Cat: M1028-100 ml)

PEI (Poly ethyleneimine) (Sigma, Cat: P3143)

Microscint 20 cocktail (PerkinElmer, Cat: 6013329)

Naltrindole (Sigma, Cat; N115)

(±)trans-U-50488 (Sigma, Cat: D8040)

DAMGO (Sigma, Cat: E7384)

Assay Buffer

Op-delta Assay Buffer Final Concentration Tris-HCl 50 mM MgCl2 10 mM EDTA  1 mM Adjust pH to 7.4, stored at 4° C.

Op-kappa Assay Buffer Final Concentration Tris-HCl 50 mM Adjust pH to 7.4, stored at 4° C.

Op-mu Assay Buffer Final Concentration Tris-HCl 50 mM MgCl2  5 mM Adjust pH to 7.4, stored at 4° C. Wash Buffer

op-kappa, op-delta and op-mu Wash Buffer Final Concentration Tris-HCl 50 mM Adjust pH to 7.4, stored at 4° C. Methods 1) Membrane and Radio Ligand Preparation

Membrane Concentration Radioligand Target (ug/well) Radio ligand concentraton(nM) DOR 6.7 [3H]-DADLE 0.5 MOR 20 [3H]DAMGO 0.5 KOR 6.7 [3H]Diprenorphine 0.3 2) Compound Preparation

Compound Ref: starting starting Ref: Final NSB conc. In conc. In Starting compound source Final Starting source Conc. In Conc. In plate Conc. In assay plate assay assay Target (mM) plate (nM) (mM) plate (nM) plate DOR 2 10000 0.02 100 naltrindole (1 μM) MOR 2 10000 0.2 1000 naltrindole (1 μM) KOR 2 10000 0.2 1000 trans-U-50488 (5 μM) 3) Assay Procedure 1) Transfer 1 μl of specified concentration compound to assay plate according to the plate map for nonspecific binding. Transfer 1 μl of DMSO to assay plate according to plate map for total binding. 2) Follow the plate map. Dispense 99 μl of membrane stocks into the plate. 3) Add 100 μl of radio ligand. 4) Seal the plates. Incubate at RT for 1 hour. 5) Soak the Unifilter-96 GF/C filter plates with 50 μl of 0.3% PEI per well for at least 0.5 hour at room temperature. 6) When binding assays are completed, filter the reaction mixture through GF/C plates using Perkin Elmer Filtermate Harvester, and then wash each plate for 4 times with cold wash buffer. 7) Dry the filter plates for 1 hour at 50 degrees. 8) After drying, seal the bottom of the filter plate wells using Perkin Elmer Unifilter-96 backing seal tape. Add 50 μl of Perkin Elmer Microscint 20 cocktail. Seal top of filter plates with Perkin Elmer TopSeal-A sealing film. 9) Count ³H trapped on filter using Perkin Elmer MicroBeta2 Reader second day. 10) Analyze the data with GraphPad Prism 5. Calculate the “Inhibition [% Control]” using the equation: % Inh=(1−Background subtracted Assay value/Background subtracted HC value)*100.

Reference KOR U-50488 DOR naltrindole MOR DAMGO

TABLE 5a DOR Compound IC50 (nM) MaxDose (nM) % Inh@MaxDose 30 1854 10000 84.3 32 >10000 10000 43.2 25 9540 10000 62 6 499.5 10000 70.9 23 106.7 10000 95.6 24 2121 10000 48 34 >10000 10000 22.7 44 190.1 10000 79.1 45 68.4 10000 73.8 46 1360 10000 72.3 nalmefene 18.1 1000 94.5 Naltrindole 0.2 100 98.8

TABLE 5b KOR Compound IC50 (nM) MaxDose (nM) % Inh@MaxDose 30 88.7 10000 97.6 32 2116 10000 80.1 25 889.3 10000 76.9 6 51.1 10000 99.6 23 11.7 10000 101.6 24 37.5 10000 82.9 34 1767 10000 68.1 44 18.4 10000 99.2 45 5.5 10000 100.1 46 33.85 10000 101.8 nalmefene 2 1000 103 U-50488 10.1 1000 101.6

TABLE 5c MOR Compound IC50 (nM) MaxDose (nM) % Inh@MaxDose 30 9 10000 82.5 32 17.4 10000 75.9 25 5 10000 87.3 6 14.9 10000 102.5 23 2.8 10000 98.7 34 12.55 10000 92.7 44 3.029 10000 83.1 45 2 10000 93.7 46 4.523 10000 102.3 24 22.4 10000 88.7 nalmefene 0.4 1000 103.5 DAMGO 1.4 1000 99.3

Example 3: Solubility Determination

A known amount of test substance (˜40 mg) was weighed into the vial, 100 μL of oil was added and heated to 60° C. and then system was slurried to reach equilibrium. More oil was added until clear solution was obtained or the solubility was <50 mg/mL. Then the clear solution was placed at room temperature (25° C.) for 24 h to confirm whether there was solid precipitation. Extra oil was added into the vial once compound precipitated out and then the system was re-equilibrated at 1000 rpm at room temperature (25° C.). Final concentration was determined by HPLC method as described below in Table 6a and 6b.

TABLE 6a Reagent Name Grade Company Lot No. Purified Water HPLC WuXiAppTec N/A Cottonseed oil N/A SIGMA 038K000G Cottonseed oil SUPER REFINED CRODA 1070292 Sesame oil HPLC CRODA 1115393 Ethanol HPLC J. T. Baker 155943 ACN HPLC Merck I0904530 735

TABLE 6b Instrument Name Model Company Serial number Water Milli-Q Direct 8 MILLIPORE PDS-PF-WPE-01 Purification Equipment Balance Mettler-Toledo Mettler-Toledo PDS-PF-BAL-08 XPR10 Stirrer C-MAG MS 10 IKA PDS-PF-ST-01 Balance Mettler-Toledo Mettler-Toledo PDS-PF-BAL-03 MX5 HPLC Shimadzu 20AB Shimadzu PDS-PF-HPLC-12 Thermomixer YQH-0623 Eppendorf PDS-PF-TM-02 The HPLC method for Compounds 6, 12-20, and 36-43 is provided in Table 7.

TABLE 7 HPLC Method 1 Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm * 4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.0 ml/min Inject volume 10 μL Analysis Time 20 min Column Temp. 40° C. Wavelength 280 nm Gradient Time Program (min) A % B % 0 80 20 9 10 90 9.01 10 90 14 80 20 20 80 20 20.01 Stop The HPLC method for Compounds 10, 21-23, 53, 55, 56, nalmefene, and naltrexone is provided in Table 8.

TABLE 8 HPLC Method 2 Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm * 4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.0 ml/min Inject volume 10 uL Analysis Time 13 min Column Temp. 40° C. Wavelength 284 nm Gradient Time Program (min) A % B % 0 95 5 9 10 90 11 10 90 11.01 95 5 13 95 5 13.01 Stop The HPLC method for Compounds 3-5, 8, 24-25, 26-34, 44-51, 54, 55, 57, 59, and 60 is provided in Table 9.

TABLE 9 HPLC Method 3 Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm * 4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.0 ml/min Inject volume 10 μL Analysis Time 13 min Column Temp. 40° C. Wavelength 280 nm Gradient Time Program (min) A % B % 0 90 10 4 35 65 25 20 80 27 10 90 27.01 90 10 30 90 10 30.01 Stop

TABLE 10 Equilibrium solubility Heating Obs. Approximate Solubility (mg/mL) in Excipient (mg/mL) Temp. Temp. Compound Castor oil Cottonseed oil Sesame oil Ethanol Water (° C.) (° C.) Approximate Solubility (mg/mL) in Excipient 36 >217 >418.7 >411.6 20-30 0.005 40 40 36 N/A >407 >408 N/A N/A 60 25 37 >226 >405.3 >408.4 75-90 0.002 40 40 37 N/A 68.5-82   80-102 N/A N/A 60 25 38 >248 >406.9 230-411 160-200 0.617 40 40 38 N/A N/A N/A N/A 0.21 60 40 39 >218   225-402.2 >400.3 150-200 0.018 40 40 39 N/A 104-139 106-141 N/A N/A 60 25  6 N/A >420.8 >402.8 120-170 0.002 40 40  6 N/A >397 >386 N/A N/A 60 25 41 N/A >407 >403 >201 0.084 40 40 41 N/A <50 <50 N/A N/A 60 25 53 N/A >403.8 >406.3 >404 0.0143 60 40 53 N/A >408.5 >401.9 N/A N/A 60 25 42 N/A <51 <52 78-93 0.245 40 40 42 N/A <26 <25.8 N/A 0.068 60 40 43 N/A <50 <50 >257 0.05 40 40 43 N/A 140-210 133-199 N/A N/A 60 40 43 (in water) N/A N/A N/A N/A 0.048 40 40 11 N/A >407 >407 >218 0.0005 40 40 11 N/A <51 <50 N/A N/A 60 25 11 after slurry in Heptane N/A N/A N/A N/A 0.0026 N/A N/A 12 N/A 100-120 70-95 65-75 0.043 40 40 12 N/A >398.5 >411.1 N/A N/A 60 40 12 N/A <51 <50 N/A N/A 60 25 12 (in water) N/A N/A N/A N/A 0.026 40 40 54 N/A <51 <50 N/A N/A 60 25 54 (in water) N/A N/A N/A N/A <0.001 40 40 13 N/A >381 >406 >214 0.002 40 40 13 N/A <50 91-121 N/A N/A 60 25 14 N/A <50 <51 69-83 0.01 40 40 14 N/A <25 <25 N/A N/A 60 40 14 (in water) N/A N/A N/A N/A 0.0056 40 40 14 after slurry in Heptane N/A N/A N/A N/A <0.0005 N/A N/A 14 after slurry in EtOAc N/A N/A N/A N/A <0.0005 N/A N/A 15 N/A <51 <52 <20 <0.0002 40 40 15 N/A >401 >404 N/A N/A 60 40 15 N/A >402.6 >396.5 N/A N/A 60 25 15 N/A N/A >403.94 N/A N/A 60 25 16 N/A 57-67 68-81 <25 0.2059 40 40 16 N/A 201-403 202-404 N/A 0.011 60 40 17 N/A >416 >416 >209 0.0008 40 40 17 N/A >408 >487 N/A N/A 60 25 18 N/A >403 >403 >209 <0.0002 40 40 18 N/A >400 >402 N/A N/A 60 25 19 N/A 67-80 103-137 27-42 0.0468 40 40 20 N/A >414 >406 157-197 <0.0002 40 40 20 N/A 68-82 80-100 N/A N/A 60 25 61 (Nalmefene) N/A <25.0 <25.5 102-137 0.025 60 40 62 (Naltrexone) N/A <26.6 <25.9 103-137 0.295 60 40 56 N/A 25-50 25-50 <40.4 0.0679 60 40 56 N/A <50 <50 N/A N/A 60 25 55 N/A >400.1 >394.8 >401.1 0.0066 60 40 55 N/A <50 <50 N/A N/A 60 25 55 (after ETOAc slurry) N/A N/A N/A N/A <0.0005 N/A N/A 22 N/A >405.9 >401.9 >397.7 0.0314 60 40 22 N/A 67-80 67-80 N/A N/A 60 25 23 N/A >411.8 >420.7 >419.3 0.0094 60 40 23 N/A >407.7 >423.9 N/A N/A 60 25 10 N/A >420.7 >404.1 >404.7 0.0006 60 40 10 N/A 100-134 <51 N/A N/A 60 25 21 N/A   101-134.8 101-134.5 50-100 0.0006 60 40 50 N/A <51 <52 N/A N/A 60 25 50 (in water) N/A N/A N/A N/A <0.001 40 40 51 N/A <50 <46 N/A N/A 60 25 51 (in water) N/A N/A N/A N/A <0.001 40 40  8* N/A <50 <52 N/A N/A 60 25  8 (in water)* N/A N/A N/A N/A 0.57 40 40 56 NMF palmitate N/A 25-50 25-50 <40.4 0.0066 60 40 56 NMF palmitate N/A <50 <50 N/A N/A 60 25 57 N/A >423.7 >417.3 N/A N/A 60 25 57 (in water) N/A N/A N/A N/A <0.001 40 40 58 N/A 101.8-203.6 99.4-198.8 N/A N/A 60 25 58 (in water) N/A N/A N/A N/A 0.0014 40 40 59 NMF dodecanoate N/A >414 >393.3 N/A N/A 60 25 59 NMF dodecanoate (in N/A N/A N/A N/A <0.001 40 40 water) 30 N/A  252.5-404.06 252.55-398.6 N/A N/A 60 25 30 (in water) N/A N/A N/A N/A <0.001 40 40 31 N/A >416.9 >413.5 N/A N/A 60 25 31 (in water) N/A N/A N/A N/A <0.001 40 40 32 N/A >395.8 >410 N/A N/A 60 25 32 (in water) N/A N/A N/A N/A 0.0328 40 40 33 N/A <50 <51 N/A N/A 60 25 33 (in water) N/A N/A N/A N/A <0.001 40 40 28 N/A <50.2 <50.6 N/A N/A 60 25 28 (in water) N/A N/A N/A N/A <0.001 40 40 24 N/A >403 338-368 N/A N/A 60 25 24 (in water) N/A N/A N/A N/A <0.001 40 40 25 N/A >414 >404 N/A N/A 60 25 25 (in water) N/A N/A N/A N/A <0.001 40 40 34 N/A >404 >405 N/A N/A 60 25 34 (in water) N/A N/A N/A N/A <0.001 40 40 44 N/A >407 208-231 N/A N/A 60 25 44 (in water) N/A N/A N/A N/A <0.001 40 40 45 N/A >402 267-288 N/A N/A 60 25 45 (in water) N/A N/A N/A N/A <0.001 40 40 46 N/A >395.5 202-269 N/A N/A 60 25 46 (in water) N/A N/A N/A N/A <0.001 40 40 29 N/A >410.2 >416.5 N/A N/A 60 25 29 (in water) N/A N/A N/A N/A <0.001 40 40 27 N/A >394.7 >399.2 N/A N/A 60 25 27 (in water) N/A N/A N/A N/A <0.001 40 40 47 N/A >405.5 >404.9 N/A N/A 60 25 47 (in water) N/A N/A N/A N/A <0.001 40 40 48 N/A >410.5 >404.8 N/A N/A 60 25 48 (in water) N/A N/A N/A N/A 0.003 40 40 26 N/A 204.3-239   <354 N/A N/A 60 25 26 (in water) N/A N/A N/A N/A <0.001 40 40 49 N/A <50 <51 N/A N/A 60 25 49 (in water) N/A N/A N/A N/A 0.028 40 40 Equilibrium Heating Obs. solubility Temp. Temp. Solubility by volumetric flask method (mg/mL) (°C.) (° C.)  8** N/A N/A >399.8 N/A N/A 60 25  8 (in water)** N/A N/A N/A N/A <0.001 40 40  3 N/A >399.12 >400.7 N/A N/A 60 25  3 (in water) N/A N/A N/A N/A <0.001 40 40  4 N/A >395.3 >400.4 N/A N/A 60 25  4 (in water) N/A N/A N/A N/A <0.001 40 40  5 N/A >401.8 >400.6 N/A N/A 60 25  5 (in water) N/A N/A N/A N/A <0.001 40 40 *Data from compound 8 initial failed batch due to non-optimized synthesis **Data from compound 8 second batch after successfully optimized synthesis

Example 4: Stability Determination of Drug Product

Compounds were resuspended in oil vehicles, stored at room temperature for the indicated time period and assessed by HPLC. Data is presented as absolute percentage loss normalized to 30 days. Nalmefene dodecanoate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202).

TABLE 11 Reagents Name Grade Company Lot No. Water HPLC WuXiAppTec N/A ACN HPLC Merck I0911030735 Cottonseed oil N/A SIGMA 038K000G Cottonseed oil SUPER REFINED CRODA 1070292 Sesame oil N/A ACROS A0377665 Sesame oil HPLC CRODA 1115393 TFA HPLC J&K LN20M33 THF HPLC MACRON 1613729801

TABLE 12 Instruments Name Model Company Serial number Water Milli-Q Direct 8 MILLIPORE PDS-PF-WPE-01 Purification Equipment Balance Mettler-Toledo Mettler-Toledo PDS-PF-BAL-05 XP6 HPLC Shimadzu 20AB Shimadzu PDS-PF-HPLC-12

TABLE 13 HPLC Method Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm * 4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.2 ml/min Inject volume 10 uL Analysis Time 30 min Column Temp. 40° C. Wavelength 280 nm Diluent THF Gradient Time Program (min) A % B % 0 90 10 4 35 65 25 20 80 27 10 90 27.01 90 10 30 90 10 30.01 Stop

TABLE 14 % Avg Purity % Avg Purity Degradation in Degradation in Cottonseed Oil Sesame Oil Compound (normalized 30 day) (normalized 30 day) 29 0.13 0.32 27 0.13 0.32 32 — 0.13 24 0.43 (0.01) 25 0.28 (0.11) 31 1.15 0.08 6 1.48 0.31 15 0.86 0.10 17 1.05 0.26 18 1.25 0.71 23 0.35 0.09 5 0.40 0.28 59 4.60 0.51 34 (0.11) 0.02 44 0.36 (0.08) 45 0.31 0.09 46 0.04 Not tested 47 0.43 Not tested 48 0.60 Not tested 3 0.04 0.07

Example 4: Stability Determination of Drug Substance

Compounds were stored at room temperature for the indicated time period and assessed by HPLC. Data is presented as absolute percentage loss normalized to 30 days. Nalmefene dodecanoate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202).

TABLE 15 Reagent Name Grade Company Lot No. Purified Water HPLC WuXiAppTec N/A ACN HPLC Merck JA056730 TFA HPLC J&K LN20M33 THF HPLC MACRON 1613729801

TABLE 16 Instrument Name Model Company Serial number Water Milli-Q Direct 8 MILLIPORE PDS-PF-WPE-01 Purification Equipment Balance Mettler-Toledo Mettler-Toledo PDS-PF-BAL-08 XPR10 Balance Mettler-Toledo Mettler-Toledo PDS-PF-BAL-03 MX5 HPLC Shimadzu 20AB Shimadzu PDS-PF-HPLC-12

TABLE 17 HPLC method for compounds 10, 11, 21, 36, and 53 HPLC Method 1 Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm * 4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.2 ml/min Inject volume 10 uL Analysis Time 32 min Column Temp. 40° C. Diluent ACN:H2O(3:1) Wavelength 280 nm Gradient Time Program (min) A % B % 0 90 10 4 40 60 25 5 95 27 5 95 27.01 90 10 32 90 10 32.01 Stop

TABLE 18 HPLC method for compounds 14, 19 and 55 HPLC Method 2 Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm * 4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.2 ml/min Inject volume 10 uL Analysis Time 32 min Column Temp. 40° C. Diluent ACN:H2O(3:1) Wavelength 280 nm Gradient Time Program (min) A % B % 0 90 10 4 35 65 25 20 80 27 10 90 27.01 90 10 30 90 10 30 Stop

TABLE 19 HPLC method for compounds 1, 3-9, 15, 17, 18, 23-27, 29, 31, 32, 34, 35, 44-48, 57, and 59 HPLC Method 3 Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm * 4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.2 ml/min Inject volume 10 uL Analysis Time 32 min Column Temp. 40° C. Diluent THF Wavelength 280 nm Gradient Time Program (min) A % B % 0 90 10 4 35 65 25 20 80 27 10 90 27.01 90 10 30 90 10 30 Stop

TABLE 20 Purity (after room % Degradation Purity temperature Normalized Compound (initial) storage) % Degradation to 30 days 36 97.70% 81.19% 16.51% 5.27% 6 99.00% 99.56% −0.56% −0.18% 53 97.00% 89.90% 7.10% 3.95% 11 98.20% 98.96% −0.76% −0.28% 14 97.01% 97.90% −0.89% −0.33% 15 98.40% 98.64% −0.24% −0.10% 17 96.30% 98.38% −2.08% −0.84% 18 98.44% 98.43% 0.01% 0.00% 19 99.70% 100.00% −0.30% −0.13% 55 98.80% 98.89% −0.09% −0.04% 23 97.30% 99.97% −2.67% −1.31% 10 97.40% 80.74% 16.66% 8.19% 21 97.60% 98.81% −1.21% −0.60% 57 99.60% 99.84% −0.24% −0.17% 59 97.41% 99.09% −1.68% −1.23% 31 98.80% 98.80% 0.00% 0.00% 32 99.00% 99.22% −0.22% −0.23% 24 99.00% 98.86% 0.14% 0.11% 25 99.00% 98.33% 0.67% 0.38% 34 98.00% 97.87% 0.13% 0.07% 44 98.00% 98.40% −0.40% −0.22% 45 98.00% 98.49% −0.49% −0.28% 46 97.00% 96.97% 0.03% 0.02% 29 99.00% 99.43% −0.43% −0.46% 27 98.90% 98.11% 0.79% 0.85% 47 99.00% 99.24% −0.24% −0.29% 48 99.00% 98.89% 0.11% 0.15% 26 99.00% 95.89% 3.11% 0.72% 7 99.00% 99.32% −0.32% −0.10% 8 98.30% 98.48% −0.18% −0.05% 9 99.00% 97.59% 1.41% 0.35% 1 99.00% 99.18% −0.18% −0.07% 3 99.00% 99.62% −0.62% −0.30% 4 99.00% 99.73% −0.73% −0.24% 5 100.00% 100.00% 0.00% 0.00% 35 100.00% 100.00% 0.00% 0.00%

Example 5: Physical Characterization of Solid State Drug Substance

The analysis of the physical characteristics of drug substances that were in a solid state was conducted using polarized light microscopy (PLM), X-ray powder diffractometer (XRPD) assessment, Differential Scanning Calorimetry (DSC) and Thermal Gravimetric Analysis (TGA). For PLM, samples were dispersed in immersion oil and were observed using an ocular lens (10×) and objective lens (20×) under crossed polarizers. For XRPD, samples were run on a diffractometer using the following method: Tube—Cu: K-alpha (λ=1.54179 Å); Generator—Voltage 40 kV, Current 40 mA; Scan scope—3 to 40°; sample rotation speed—15 rpm; scanning rate—10 deg/min. For DSC, ˜1 mg of sample was tested using a crimped aluminum pan and covered by a lid with a hole, heated from room temperature to 300° C. at a speed of 10° C./minute. For TGA, 2-5 mg of sample was placed in an open platinum pan and heated from room temperature to 300° C. at a rate of 10° C./minute. Nalmefene palmitate were previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202).

TABLE 21 XRPD, TGA and DSC results X-ray Powder Thermal Gravimetric Differential scanning Cpd # Diffraction Analysis calorimetry 11 Crystal Form with obvious Weight loss of 0.30% at Single endothermic peak birefringence 120° C. following at 56.71° C. which could be decomposition melting point. 12 Crystal Form with obvious Weight loss of 0.19% at Single endothermic peak birefringence 120° C. following at 51.94° C. which could be decomposition melting point. 14 Weak crystallinity of the Weight loss of 1.649% at Single endothermic peak compound 120° C. following at 124.66° C. which could decomposition be melting point. 16 Crystal Form with obvious Weight loss of 0.403% at Single endothermic peak birefringence 120° C. following at 59.04° C. which could be decomposition melting point. 19 Crystal Form with obvious Weight loss of 0.1623% at Single endothermic peak birefringence 120° C. following at 56.31° C. which could be decomposition melting point. 56 Crystal Form with obvious Weight loss of 0.1815% at Two endothermic peak at birefringence 120° C. following 48.95° C. and 59.75° C. decomposition 55 Crystal Form with obvious Weight loss of 0.2597% at Single endothermic peak birefringence 120° C. following at 48.24° C. which could be decomposition melting point. 10 Crystal Form with obvious Weight loss of 0.3047% at Single endothermic peak birefringence 120° C. following at 47.18° C. which could be decomposition melting point. 40 Crystal form with partial Weight loss of 0.57% at Two endothermic peak at birefringence 120° C. following 107.6° C. and 195.0° C. decomposition 21 Crystal Form with partial Weight loss of 1.133% at Single endothermic peak birefringence. 120° C. following at 70.84° C. which could be decomposition melting point. 31 Crystal Form with obvious Weight loss of 0.7244% at Single endothermic peak birefringence 120° C. following at 45.15° C. which could be decomposition melting point.

Example 5: Polymorph Screening of Solid State Drug Substances

In order to identify stable polymorph forms of solid state drug substances, approximately 50 mg of compound (nalmefene or naltrexone equivalnets) was weighed into vials. Next, 500 μL of the indicated solvents was added and the suspension was stirred at 700 rpm, 50° C. for 72 hours. For samples in suspension, solids were separated by centrifuge (10 minutes, 14000 rpm) and dried in vacuum oven at 30° C. overnight. For samples in solution, solids were generated by evaporation (stir bar removed and covered with aluminum foil with pinholes, then dried in vacuum oven at 30° C. overnight). Dried solids were characterized by XRPD, TGA and DSC. Results are presented in Table 23.

TABLE 23 Target XRPD conc. Visual observation results Compound # Solvents (mg/ml) RT 50° C. Dry method and appearance (Dried) Comment 11 Heptane 100 Clear Clear Evaporation/white powder Pattern B Initial crystal form (pattern A) likely MTBE Clear Clear Evaporation/white powder Pattern A to be stable form; Pattern A generated Isopropanol Slurry Clear Evaporation/white powder Pattern A by evaporation with MTBE, IPA, EtOAc Slurry Clear Evaporation/white powder Pattern A EtOAc and Acetone, Compound Acetone Slurry Clear Evaporation/white powder Pattern A evaporated in heptane was different form 14 Heptane 100 Slurry Slurry Centrifugation/white powder Pattern B Initial crystal form (pattern A) MTBE Clear Clear Evaporation/white powder Pattern A appears unstable; Same crystal from Isopropanol Clear Clear Evaporation/white powder Pattern A generated by evaporation from EtOAc Slurry Clear Evaporation/white powder Pattern A MTBE, IPA, EtOAc and acetone and Acetone Slurry Clear Evaporation/white powder Pattern A might be unstable. Form evaporated in heptane different form and likely more stable 16 Heptane 100 Slurry Slurry Centrifugation/white powder Pattern A Initial crystal form (pattern A) is MTBE Clear Clear Evaporation/white powder Pattern A likely stable form. Same crystal form Isopropanol Slurry Slurry Centrifugation/white powder Pattern A (pattern A) was generated by EtOAc Slurry Clear Evaporation/white powder Pattern A evaporation from MTBE, EtOAc and Acetone Slurry Clear Evaporation/white powder Pattern A Acetone. Same crystal form (pattern A) generated by slurry in Heptane and IPA 19 Heptane 100 Slurry Clear Evaporation/white powder Pattern A Initial crystal form (pattern A) likely MTBE Clear Clear Evaporation/white powder Pattern A to be stable fomr. Same crystal form Isopropanol Slurry Clear Evaporation/white powder Pattern A (pattern A) was generated by EtOAc Slurry Clear Evaporation/white powder Pattern A evaporation from Heptane, MTBE, Acetone Slurry Clear Evaporation/white powder Pattern A IPA, EtOAc and Acetone 55 Heptane 100 Slurry Clear Evaporation/white powder Pattern B Cannot determine which crystal form MTBE Clear Clear Evaporation/white powder Pattern B is more stable. Same form (pattern B) Isopropanol Slurry Clear Evaporation/white powder Pattern B was generated by evaporation from EtOAc Slurry Clear Evaporation/white powder Pattern B Heptane, MTBE, IPA, EtOAc and Acetone Slurry Clear Evaporation/white powder Pattern B Acetone. 10 Heptane 100 Slurry Slurry Centrifugation/White wax Pattern A Initial crystal form (pattern A) is a MTBE Clear Clear Evaporation/White wax Pattern A stable form. Same form (pattern A) Isopropanol Slurry Clear Evaporation/White wax Pattern A generated by evaporation from EtOAc Slurry Clear Evaporation/White wax Pattern A MTBE, IPA, EtOAc and Acetone. Acetone Slurry Clear Evaporation/White wax Pattern A Pattern A also generated by slurry in heptane. 21 Heptane 100 Slurry Clear Evaporation/white powder Pattern A Initial crystal form (pattern A) is a MTBE Clear Clear Evaporation/white powder Pattern A stable form. Same crystal form Isopropanol Slurry Clear Evaporation/white powder Pattern A (pattern A) was generated by EtOAc Slurry Clear Evaporation/white powder Pattern A evaporation from Heptane, MTBE, Acetone Slurry Clear Evaporation/white powder Pattern A IPA, EtOAc and Acetone 31 Heptane 100 Slurry Clear Evaporation/white powder Pattern A Initial crystal form (pattern A) very MTBE Slurry Clear Evaporation/white powder Pattern A likely to be a stable form. Same Isopropanol Clear Clear Evaporation/white powder Pattern A crystal form (pattern A) was generated EtOAc Clear Clear Evaporation/white powder Pattern A by evaporation from MTBE, IPA, Acetone Slurry Clear Evaporation/white powder Pattern A EtOAc and acetone 40 Heptane 100 Slurry Slurry Centrifugation/white powder Pattern A Initial crystal form (pattern A) is a MTBE Clear Clear Evaporation/white powder Amorphous stable form. Same crystal form Isopropanol Slurry Clear Evaporation/oil Amorphous (Pattern A) was generated by slurry in EtOAc Slurry Clear Evaporation/white powder Pattern A heptane and evaporation form EtOAc. Acetone Slurry Clear Evaporation/white powder Amorphous Compound became amorphous by evaporation from MTBE, IPA and acetone. III. Preparation of Pharmaceutical Dosage Forms

Example 1: Oral Capsule

The active ingredient is a compound of Table 1, or a pharmaceutically acceptable salt thereof. A capsule for oral administration is prepared by mixing 1-1000 mg of active ingredient with starch or other suitable powder blend. The mixture is incorporated into an oral dosage unit such as a hard gelatin capsule, which is suitable for oral administration.

Example 2: Solution for Injection

The active ingredient is a compound of Table 1, or a pharmaceutically acceptable salt thereof, and is formulated as a solution in sesame oil, cottonseed oil, castor oil or other pharmaceutically acceptable lipophilic excipient, preferably at a concentration of greater than 100 mg/mL. The resulting solution is administered by intramuscular injection.

Compounds were resuspended to 1 mL at the indicated concentrations (in mg/ml base equivalents) by mixing with magnetic stirring (1000 rpm) at 60° C. until a homogeneous clear solution was achieved, then cooled down to room temperature and stored protected from light. Appearance of oil formulations was observed and recorded at room temperature (25° C.) at initial, 2 hours, and 24 hours. Samples for “Assay” measurements were taken at initial, 2 hours and 24 hours post resuspension and subjected to HPLC analysis where actual concentration was based on a standard curve (Assay=Concentration(measured by HPLC)/Actual concentration(by weight)×100%). Purity was calculated at indicated time points based on the percentage of area under the curve of the main peak from the HPLC spectrogram. Syringability was assessed by drawing through a 21 Gauge needle. Some indicated samples were assessed for Appearance, Assay and Purity after 7 months in 40° C./75% Relative Humidity. Data are presented in Table 21.

TABLE 24 Target Conc. Syringability Follow up (mg/ml) in Appearance Assay Purity (21G needle) observation Base 24 2 24 2 24 24 at room Compound Vehicle Volume eq. Initial 2 hours hours Initial hours hours Initial hours hours Initial 2 hours hours temperature 6 Sesame 1 mL 200 Light Light Light 102.26% 102.32% 99.30% 99.48% 99.46% 99.37% feasible feasible feasible Remains in oil + 10 amber amber amber solution uL transparent transparent transparent Benzyl oil oil oil Alcohol Sesame 1 mL 300 Light Light Light 99.82% 99.66% 102.90% 99.37% 99.35% 99.44% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 400 Light Light Light 98.06% 97.94% 98.43% 99.37% 99.39% 99.37% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil 15 Sesame 1 mL 200 Light Light Light 107.68% 109.70% 109.70% 96.38% 96.44% 96.34% feasible feasible feasible Remains in oil + 10 amber amber amber solution uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 300 Light Light Light 101.55% 108.28% 103.00% 96.44% 96.38% 96.45% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 400 Light Light Light 93.52% 99.92% 94.15% 96.42% 96.28% 96.41% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil 17 Sesame 1 mL 200 Light Light Light 98.80% 98.66% 98.29% 98.61% 98.64% 98.61% feasible feasible feasible Remains in oil + 10 amber amber amber solution uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 300 Light Light Light 105.43% 105.79% 105.65% 98.61% 98.60% 98.63% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 400 Light Light Light 98.51% 98.82% 97.58% 98.63% 98.60% 98.59% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil 18 Sesame 1 mL 200 Light Light Light 97.56% 97.79% 97.85% 99.15% 99.05% 99.07% feasible feasible feasible Remains in oil + 10 amber amber amber solution uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 300 Light Light Light 99.00% 100.43% 99.52% 98.93% 99.06% 98.84% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 400 Light Light Light 98.54% 98.70% 98.77% 99.17% 99.13% 99.14% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil 23 Sesame 1 mL 200 Light Light Light 97.22% 97.54% 97.10% 99.50% 99.48% 99.47% feasible feasible feasible Remains in oil + 10 amber amber amber solution uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 300 Light Light Light 100.34% 100.25% 99.51% 99.49% 99.48% 99.50% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 400 Light Light Light 97.54% 97.92% 95.81% 99.51% 99.48% 99.49% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil 57 Sesame 1 mL 200 Light Light Light 104.75% 104.82% 104.02% 99.55% 99.49% 99.54% feasible feasible feasible Insoluble: oil + 10 amber amber amber gross uL Benzyl transparent transparent transparent precipitant/ Alcohol oil oil oil phase Sesame 1 mL 300 Light Light Light 102.67% 102.47% 100.66% 99.69% 99.67% 99.65% feasible feasible feasible seperation oil + 10 amber amber amber after 23 days uL Benzyl transparent transparent transparent Alcohol oil oil oil Sesame 1 mL 400 Light Light Light 101.03% 101.18% 100.34% 99.63% 99.59% 99.63% feasible feasible feasible oil + 10 amber amber amber uL Benzyl transparent transparent transparent Alcohol oil oil oil 31 Sesame 1 mL 200 Light Light Light 101.90% 100.35% N/A 99.29% 99.28% N/A feasible feasible feasible Insoluble: oil + 10 amber amber amber gross uL Benzyl transparent transparent transparent precipitant/ Alcohol oil oil oil phase Sesame 1 mL 300 Light Light White 105.84% 95.68% N/A 99.14% 99.31% N/A feasible feasible N/A seperation oil + 10 amber amber wax after 24 hr uL Benzyl transparent transparent Alcohol oil oil Sesame 1 mL 400 Light Light White 99.16% 95.81% N/A 99.23% 99.25% N/A feasible feasible N/A oil + 10 amber amber wax uL Benzyl transparent transparent Alcohol oil oil 32 Sesame 1 mL 300 Light Light Light 95.61% 96.59% 95.42% 98.78% 98.63% 0.9878 feasible feasible feasible Insoluble: oil + 10 amber amber amber gross uL Benzyl transparent transparent transparent precipitant/ Alcohol oil oil oil phase Sesame 1 mL 350 Light Light White 102.17% 103.30% N/A 98.79% 98.76% N/A feasible feasible N/A seperation oil + 10 amber amber wax after 24 hr uL Benzyl transparent transparent Alcohol oil oil Sesame 1 mL 400 Light Light White 97.21% 97.10% N/A 98.80% 98.78% N/A feasible feasible N/A oil + 10 amber amber Wax uL Benzyl transparent transparent Alcohol oil oil 24 Cottonssed 1 mL 400 Light Light Light 107.62% 99.58% 100.58% 99.02% 98.86% 98.93% feasible feasible feasible Remains in oil + 10 clear clear clear solution uL Benzyl yellow yellow yellow Alcohol oil oil oil 29 Sesame 1 mL 400 Light Light Light 103.07% 104.83% 103.35% 99.21% 99.21% 99.28% feasible feasible feasible Drug oil + 10 amber amber amber substance uL Benzyl transparent transparent transparent shown to be Alcohol oil oil oil unstable in oil solution at 4 months 40 C./75% RH 27 Sesame 1 mL 400 Light Light Light 95.52% 98.43% 96.46% 99.33% 99.30% 99.09% feasible feasible feasible Insoluble: oil + 10 amber amber amber gross uL Benzyl transparent transparent transparent precipitant/ Alcohol oil oil oil phase seperation after 90 days 47 Sesame 1 mL 400 Light Light Light 102.41% 101.60% 101.72% 99.37% 99.31% 99.26% feasible feasible feasible Insoluble: oil + 10 amber amber amber gross uL Benzyl transparent transparent transparent precipitant/ Alcohol oil oil oil phase seperation after 24 hr

TABLE 25A Target conc. (mg/ml) Appearance Assay Sample Nalmefene 7 months in 7 months in 7 months in 7 months in Compound # Vehicle Volume amount Initial 2 hours 24 hours 40 C./75% RH Initial 2 hours 24 hours 40 C./75% RH-1 40 C./75% RH-2 40 C./75% RH-3 25 Sesame 1 + 1 mL 400 Light Light Light Light hazy 102.48% 102.90% 108.25% 98.02% 94.38% 98.31% oil + 20 uL amber amber amber oil, Benzyl transparent transparent transparent possible Alcohol oil oil oil immiscible particles. 34 Sesame 1 + 1 mL 400 Light Light Light Yellow 101.77% 102.07% 101.11% 102.01% 99.07% 97.99% oil + 20 uL amber amber amber hazy oil, Benzyl transparent transparent transparent possible Alcohol oil oil oil immiscible oil droplets. 44 J&K    1 mL 350 Light Light Light 101.41% 102.44% 102.32% Cottonseed clear clear clear oil + 10 yellow yellow yellow uL oil oil oil Benzyl Alcohol 45 J&K 1 + 1 mL 300 Light Light Light Yellow 98.86% 98.68% 98.64% 100.57% 99.54% 98.78% Cottonseed yellow yellow yellow hazy oil, oil + 20 clear oil clear oil clear oil possible uL small oil Benzyl droplets Alcohol 46 J&K 1 + 1 mL 300 Light Light Light Yellow 98.94% 99.28% 99.00% 106.24% 108.43% 107.85% Cottonseed yellow yellow yellow hazy non- oil + 20 hazy oil hazy oil hazy oil homogenous uL oil with Benzyl obvious Alcohol immiscible oil droplets 48 Sesame    1 mL 400 Light Light Light 97.50% 96.63% 96.39% oil + 10 uL hazy oil hazy oil hazy oil Benzyl Alcohol

TABLE 25B Purity 7 7 7 months months months Syringeability Follow-up Com- in 40 C./ in 40 C./ in 40 C./ (21 Gauge needle) Follow up observation pound 75% 75% 75% 2 24 observation at 7 months at # Initial 2 hours 24 hours RH-1 RH-2 RH-3 Initial hours hours at room temp 40 C./75% RH 25 98.36% 98.64% 98.57% 97.07% 97.13% 97.07% fea- fea- fea- Remains in No clearly sible sible sible solution visible particles on PLM microcopy 34 98.59% 98.58% 98.50% 99.35% 99.44% 99.46% fea- fea- fea- Remains in No clearly sible sible sible solution visible particles on PLM microcopy 44 98.68% 98.41% 98.73% fea- fea- fea- Insoluble: sible sible sible grossly hazy with visible solid precipitate after 20 days 45 99.58% 101.17%  100.62%  94.60% 94.68% 94.67% fea- fea- fea- Remains in Possible sible sible sible solution small visible particles on PLM microcopy 46 97.96% 97.96% 97.95% 95.48% 95.42% 95.54% fea- fea- fea- Insoluble: grossly Obvious sible sible sible hazy with visible particles on solid precipitate on PLM after 20 days microscopy 48 98.97% 98.80% 98.53% fea- fea- fea- Insoluble: grossly sible sible sible hazy with visible solid precipitate after 170 days. Crystalline particles on PLM microscopy. Drug substance changed from colorless oil to white wax within 4 weeks.

TABLE 26A Target conc. Appearance Com- (mg/ml) 3 months in 6 months in pound Sample Nalmefene 40 C./75% 40 C./75% # Vehicle Volume amount Initial 24 hours RH RH 6 Sesame oil + 50 uL 5 mL 400 Light amber Light amber Light amber Light amber Benzyl Alcohol transparent transparent transparent transparent oil oil oil (at 25 C.) oil 15 Sesame oil + 50 uL 5 mL 400 Light amber Light amber Light amber Light amber Benzyl Alcohol transparent transparent transparent transparent oil oil oil oil 17 Sesame oil + 50 uL 5 mL 400 Light amber Light amber Light amber Light amber Benzyl Alcohol transparent transparent transparent transparent oil oil oil oil 18 Sesame oil + 50 uL 5 mL 400 Light amber Light amber Light amber Light amber Benzyl Alcohol transpoilarent transparent transparent transparent oil oil oil oil 9 Sesame oil + 50 uL 5 mL 400 Light amber Light amber Light amber Light amber Benzyl Alcohol transparent transparent transparent transparent oil oil oil oil 59 Sesame oil + 10 uL 5 mL  86 Light amber Light amber Benzyl Alcohol transparent transparent oil oil 24 Cottonseed oil + 200 20 mL  400 Pale yellow uL Benzyl Alcohol oil 45 Sesame oil + 1% 20 mL  400 clear oil Benzyl alcohol Sesame oil + 1% 1 mL 400 clear oil Benzyl alcohol Sesame oil + 1% 1 mL 400 clear oil Benzyl alcohol Sesame oil + 1% 1 mL 400 clear oil Benzyl alcohol 34 Sesame oil + 100 ul 10 mL  400 Brown oil Benzyl Alcohol 7 Sesame oil + 1% 5 + 1 + 1 + 400 Almost clear Benzyl alcohol 1 mL solution 43 Sesame oil + 1% 10 mL  400 Brown oil Benzyl alcohol 9 Sesame oil + 1% 5 ml 400 Almost clear Benzyl alcohol 1 ml solution 1 ml 1 ml 1 Sesame oil + 1% 5 + 1 + 400 Slightly turbid Benzyl alcohol 1 mL oil 3 Sesame oil + 1% 5 + 5 mL 400 Light amber Light amber Benzyl alcohol transparent oil transparent oil (6 hr) 4 Sesame oil + 1% 5 + 1 + 400 Almost clear Benzyl alcohol 1 mL solution 5 Sesame oil + 1% 1 + 1 + 400 Light amber Benzyl alcohol 1 mL transparent oil 35 Sesame oil + 1% 10 mL  400 Almost Benzyl alcohol clear oil

TABLE 26B Assay 3 months 3 months 3 months 6 6 6 in in in months in months in months in Compound Initial - Initial - Initial - 24 hr - 24 hr - 24 hr - 40 C./75% 40 C./75% 40 C./75% 40 C./75% 40 C./75% 40 C./75% # 1 2 3 l 2 3 RH-1 RH-2 RH-3 RH-1 RH-2 RH-3 6 103.19% 106.47% 104.72% 100.81% 100.60% 101.40% 102.15% 101.9%  99.07% 101.25% 101.08% 101.24% (25 C.) (25 C.) (25 C.) 15  99.37%  98.69% 101.17%  97.06% 101.32%  99.43%  97.75%  97.98%  97.53% 101.91% 101.82% 102.09% 17  98.84%  99.62%  99.49%  98.02%  98.22%  98.07% 101.42% 101.39% 101.30% 103.37% 103.24% 103.18% 18  93.67%  98.55%  96.11%  95.75%  95.77%  93.03% 102.46% 103.29% 102.94%  99.73% 100.49% 100.23% 9 102.27% 100.89% 101.56%  99.70%  97.28%  90.59% 101.74% 102.03% 101.57%  98.23%  98.80%  93.52% 59 102.52% 102.59% 102.71% 103.05% 102.47% 102.90% 24 112.73% 105.24% 103.76% 45  98.12%  98.99% 100.30% 100.05% 101.02%  99.92%  99.86% 101.14%  99.39%  99.91%  99.03%  99.99% 34  96.78%  97.68%  98.16% 7  99.73% 100.16% 101.37% 43  99.27%  99.39%  97.86% 9  99.23%  99.79%  99.83%  99.35%  98.76%  98.66%  98.93% 100.93% 103.17% 109.00% 109.82% 111.59% 1 110.88% 111.97% 110.59% 3 N/A N/A N/A  97.91%  97.55%  94.13% (6 days) (6 days) (6 days) 4 100.31%  97.12%  97.52% 5  96.64%  95.72%  96.29% 35  98.21%  96.79%  99.37%

TABLE 26C Purity 3 3 3 6 6 6 months in months in months in months in months in months in Syringeability Follow up 40 C./ 40 C./ 40 C./ 40 C./ 40 C./ 40 C./ (21 Gauge needle) observation Compound 24 24 75% 75% 75% 75% 75% 75% 24 3 6 at # Initial - 1 Initial - 2 Initial - 3 24 hr - l hr - 2 hr - 3 RH-1 RH-2 RH-3 RH-1 RH-2 RH-3 Initial hours months months room temp 6 99.48% 99.46% 99.37% 99.36% 99.35% 99.44% 99.29% 99.81% 98.75% 98.88% 98.86% 98.89% feasible feasible feasible feasible Remains in (25 C.) (25 C.) (25 C.) Solution 15 96.87% 97.19% 96.88% 96.80% 96.39% 96.84% 96.86% 96.57% 96.70% 96.20% 96.18% 96.21% feasible feasible feasible feasible Remains in Solution 17 98.49% 98.70% 98.78% 98.45% 98.65% 98.82% 98.52% 98.29% 98.92% 98.74% 98.75% 98.70% feasible feasible feasible feasible Remains in (25 C.) (25 C.) (25 C.) Solution 18 99.32% 99.09% 98.97% 99.00% 99.14% 99.07% 98.38% 98.39% 98.39% 98.94% 98.54% 98.71% feasible feasible feasible feasible Remains in Solution 9 99.38% 99.36% 99.49% 99.33% 99.56% 99.44% 99.44% 99.17% 99.49% 99.56% 99.53% 99.23% feasible feasible feasible feasible Remains in Solution 59 99.00% 98.88% 98.94% 99.03% 98.87% 98.91% feasible feasible Remains in Solution 24 99.63% 99.62% 99.64% feasible Remains in Solution 45 99.73% 99.71% 99.68% feasible Remains in 99.48% 99.38% 99.58% N/A Solution 99.86% 99.63% 99.68% N/A 99.73% 99.57% 99.78% N/A 34 95.93% 95.84% 95.91% feasible Purity of compound in Drug product was 95.9%. Was found to be 99% after synthesis. Precipitation at 55 days 7 99.34% 99.31% 99.23% feasible Remains in Solution 43 98.60% 98.61% 98.49% feasible Remains in Solution 9 98.26% 98.14% 97.98% feasible Hazy homogenous appearance, possible solution ~24hr Visible sold precipitant ~24 hr Visible sold precipitant ~24 hr Clearly precipitated out of solution ~24 hr 1 100.00%  100.00%  100.00%  feasible Remains in Solution 3 N/A N/A N/A  99.5% 99.54% 99.34% feasible Remains in (6 days) (6 days) (6 days) Solution 4 99.65% 99.66% 99.76% feasible Remains in Solution 5 99.72% 99.71% 99.72% feasible Remains in Solution 35 100.00%  100.00%  100.00%  feasible Remains in Solution IV. Pharmacokinetic Evaluation

Example 1: Rat Pharmacokinetic Studies

Purpose

The purpose of this study is to determine the pharmacokinetics of test compounds in plasma, following intramuscular administration to male Sprague Dawley Rats (n=3, unless otherwise specified).

Acclimation/Quarantine

Animals are assessed as to their general health and acclimated for at least 3 days before being placed on study.

Animal Husbandry

Animals are housed during acclimation and individually housed during the study. The animal room environment was controlled (target conditions: temperature 18 to 26° C., relative humidity 30 to 70%, 12 hours artificial light and 12 hours dark). Temperature and relative humidity were monitored daily. Water was provided to the animals ad libitum.

Animal Body Weights and Clinical Observation

Body weights were determined before selection to the study and on the day of dose administration. Weight monitoring was done every week.

Detailed clinical observation including behavior and activity, reflection, respiration, skin and fur, facial feature, genitourinary system, and other gross lesions was performed on the dosing day and at each sample collection time point.

Dose Administration

The dose formulation of 400 mg base equivalents/ml in sesame oil+1% benzyl alcohol (unless otherwise specified) was administered by intramuscular injection. The dose volume was determined by the animals' body weight determined on the morning of dosing day.

Sample Collection

Each blood collection (about 0.2 mL per time point) was performed from jugular vein puncture of each animal into pre-chilled plastic microcentrifuge tubes containing 5 μL of 160 mg/mL sodium fluoride/potassium oxalate (NaF/KO=1/3) with 5% PMSF(100 mM in ethanol) as stabilizer and 4 μL of EDTA-K2 as anti-coagulant and placed on wet ice until centrifugation.

Plasma Processing

Each collected blood sample was centrifuged for 4 minutes at 4° C. and 10000 rpm for plasma collection. Plasma was collected and transferred into a pre-labeled PP tube in dry ice at each time point and precipitated immediately using ACN at a ratio of 1:4 (plasma:ACN). Centrifuged again (10 minutes, 12000 rpm) and obtain the supernatant.

After terminal collection, all supernatant was stored at approximately −80° C. until bioanalysis.

Bioanalytical Method and Sample Analysis

LC-MS/MS methods for the quantitative determination of test compound in biological matrix were developed. A calibration curve with 8 non-zero calibration standards were applied for the method including LLOQ (0.05 ng/ml). The sample analysis was performed concurrently with a set of calibration standards and two sets of QC samples using the LC-MS/MS method.

Data Analysis

Plasma concentration versus time data was analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C_(max), T_(max), T_(1/2), AUC_((0-t)), AUC_((0-inf)), MRT_((0-t)), MRT_((0-inf)) and graphs of plasma concentration versus time profile were prepared.

The dose for nalmefene dodecanoate was determined by allometric scaling to rat from dog doses as previously reported (Gaekens et al, Journal of Controlled Release 232 (2016) 196-202). Terminal half life was determined for active metabolite of select compounds, and is used for estimating duration above minimally effective plasma concentration for the active metabolite.

TABLE 27 Nominal Half-life t_(1/2) Terminal AUC_(0-inf) AUC_(0-inf) Dose (hour) Half-life t_(1/2) (ng · h/mL) (ng · h/mL) Compound (mg/kg) Vehicle prodrug (hour) nalmefene prodrug nalmefene Nalmefene 0.80 saline — 0.87 — 66.0 HCl-IR 59 17 Sesame oil + 1% 15.2 248 10.4 1026 Benzyl Alcohol 6 80 Sesame oil + 1% 340 569 78.9 3576 Benzyl Alcohol 6 123 Sesame oil + 1% 213 425 620 14704 Benzyl Alcohol 6 165 Sesame oil + 1% 204 491 637 18876 Benzyl Alcohol 15 80 Sesame oil + 1% 1553 639 679 3046 Benzyl Alcohol 15 123 Sesame oil + 1% 884 993 2574 7232 Benzyl Alcohol 15 165 Sesame oil + 1% 500 451 2789 14019 Benzyl Alcohol 17 200 Sesame oil + 1% Prodrug not 266 Prodrug not 16178 Benzyl Alcohol detected detected 18 80 Sesame oil + 1% Prodrug not 599 Prodrug not 8217 Benzyl Alcohol detected detected 18 123 Sesame oil + 1% Prodrug not 3409 Prodrug not 13658 Benzyl Alcohol detected detected 18 200 Sesame oil + 1% Prodrug not 847 Prodrug not 15104 Benzyl Alcohol detected detected 23 200 Sesame oil + 1% Prodrug not Not Prodrug not 27105 (naltrexone Benzyl Alcohol detected assessed detected (naltrexone metabolite) metabolite) 24 80 Cottonseed + 1% 398 1093 147 5584 Benzyl Alcohol 24 123 Cottonseed 717 445 547 11283 oil + 1% Benzyl Alcohol 24 165 Cottonseed 851 458 453 19031 oil + 1% Benzyl Alcohol 29 165 Sesame oil + 1% 175 911 74.8 17313 Benzyl Alcohol (naltrexone metabolite) No adverse affect on body weight or clinical observations were noted in any rats across all studies.

Time vs nalmefene concentration data for nalmefene HCL in 1 mg/ml at 0.80 mg/kg is provided in Table 28a.

TABLE 28a Mean conc Time (h) Nalmefene (ng/mL) 1.00 29.4 2.00 2.09 4.00 0.212 8.00 0.0178 12 ND* 24.0 ND *Not detected *Not detected

Time vs nalmefene concentration data for compound 59 (nalmefene dodecanoate) in 86 mg/ml concentration at 17 mg/kg is provided in Table 28b.

TABLE 28b Mean conc Time (h) Nalmefene (ng/mL) 1.00 7.85 2.00 8.10 4.00 7.45 8.00 6.85 24.0 6.96 48.0 7.92 72.0 5.77 144 1.59 312 0.403 480 0.235 648 0.145

Time vs nalmefene concentration data for compound 6 at 80 mg/kg is provided in Table 29.

TABLE 29 Mean conc Time (h) Nalmefene (ng/mL) 1.00 9.51 2.00 7.11 4.00 5.48 8.00 5.09 24.0 5.74 48.0 8.48 72.0 7.18 144 3.73 312 3.08 480 2.49 648 2.09 816 1.80 984 1.39 1152 1.14 1320 0.833 1488 0.413

Time vs nalmefene concentration data for compound 6 at 123 mg/kg is provided in Table 30.

TABLE 30 Mean conc Time (h) Nalmefene (ng/mL) 0.250 43.8 1.00 36.3 2.00 24.0 4.00 16.7 8.00 12.1 24.0 11.0 48.0 14.2 72.0 10.7 144 9.17 312 13.6 480 10.6 648 11.1 816 8.8 984 6.5 1152 4.0 1320 3.2 1488 1.6 1656 1.7 1824 1.2 1992 0.9 2160 0.8 2328 0.8 2496 0.6 2664 0.5

Time vs nalmefene concentration data for compound 6 at 165 mg/kg is provided in Table 31.

TABLE 31 Mean conc Time (h) Nalmefene (ng/mL) 0.250 50.4 1.00 48.0 2.00 29.2 4.00 19.3 8.00 13.9 24.0 12.3 48.0 14.4 72.0 12.4 144 10.7 312 14.5 480 18.1 648 17.1 816 14.2 984 10.5 1152 6.2 1320 4.9 1488 2.8 1656 2.3 1824 1.8 1992 1.3 2160 1.3 2328 1.1 2496 0.9 2664 0.7

Time vs nalmefene concentration data for compound 15 at 80 mg/kg is provided in Table 32.

TABLE 32 Mean conc Time (h) Nalmefene (ng/mL) 1.00 7.07 2.00 4.21 4.00 2.10 8.00 1.42 24.0 1.98 48.0 2.39 72.0 3.25 144 2.32 312 2.18 480 3.03 648 2.15 816 0.944 984 0.714 1152 0.745 1320 0.663 1488 0.706 1656 0.697 1824 0.514 1992 0.322 2160 0.441

Time vs nalmefene concentration data for compound 15 at 123 mg/kg is provided in Table 33.

TABLE 33 Mean conc Time (h) Nalmefene (ng/mL) 0.250 18.7 1.00 20.4 2.00 13.4 4.00 6.20 8.00 2.74 24.0 1.54 48.0 1.89 72.0 2.00 144 2.97 312 5.64 480 7.88 648 6.60 816 5.33 984 3.87 1152 2.41 1320 2.10 1488 2.33 1656 2.01 1824 1.93 1992 1.50 2160 1.25 2328 1.01 2496 1.18 2664 0.831

Time vs nalmefene concentration data for compound 15 at 165 mg/kg is provided in Table 34.

TABLE 34 Mean conc Time (h) Nalmefene (ng/mL) 0.250 30.3 1.00 24.1 2.00 15.3 4.00 7.60 8.00 2.92 24.0 1.65 48.0 2.43 72.0 2.60 144 3.11 312 6.45 480 13.2 648 12.5 816 7.92 984 6.30 1152 5.30 1320 4.54 1488 3.62 1656 3.24 1824 3.38 1992 2.16 2160 1.62 2328 0.966 2496 0.831 2664 0.903

Time vs nalmefene concentration data for compound 17 at 200 mg/kg is provided in Table 35.

TABLE 35 Mean conc IM Time (h) Nalmefene (ng/mL) 1.00 249 2.00 299 4.00 364 8.00 340 24.0 157 48.0 80.0 72.0 44.3 144 15.5 312 5.09 480 3.04 648 3.37 816 1.90 984 1.29 1152 0.737 1320 0.375 1488 0.309

Time vs nalmefene concentration data for compound 18 at 80 mg/kg is provided in Table 36.

TABLE 36 Mean conc Time (h) Nalmefene (ng/mL) 0.250 5.13 1.00 14.9 2.00 22.9 4.00 39.9 8.00 35.6 24.0 36.6 48.0 32.7 72.0 28.2 144 21.5 312 5.60 480 2.99 648 1.58 816 1.29 984 1.34 1152 0.99 1320 0.75 1488 0.505 1656 0.465 1824 0.412 1992 0.383

Time vs nalmefene concentration data for compound 18 at 123 mg/kg is provided in Table 37.

TABLE 37 Mean conc Time (h) Nalmefene (ng/mL) 0.250 3.98 1.00 15.6 2.00 24.4 4.00 43.5 8.00 43.7 24.0 39.2 48.0 29.8 72.0 26.5 144 15.9 312 4.21 480 5.59 648 4.73 816 4.01 984 4.26 1152 3.14 1320 3.10 1488 2.44 1656 2.55 1824 2.55 1992 1.38 2160 2.52 2328 3.11 2496 1.92 2664 2.27

Time vs nalmefene concentration data for compound 18 at 200 mg/kg is provided in Table 38.

TABLE 38 Mean conc Time (h) Nalmefene (ng/mL) 1.00 32.3 2.00 59.0 4.00 64.8 8.00 76.8 24.0 54.2 48.0 46.7 72.0 38.4 144 29.0 312 13.4 480 5.17 648 5.51 816 3.54 984 2.90 1152 2.80 1320 2.05 1488 1.77 1656 1.40 1824 1.68 1992 1.18 2160 1.42 2328 1.43 2496 0.781 2664 1.18 2832 2.34

Time vs naltrexone concentration data for compound 23 at 200 mg/kg is provided in Table 39.

TABLE 39 Mean conc Time (h) Naltrexone (ng/mL) 1.00 32.3 2.00 655 4.00 727 8.00 603 24.0 384 48.0 181 72.0 109

Time vs nalmefene concentration data for compound 24 at 80 mg/kg is provided in Table 40.

TABLE 40 Mean conc Time (h) Nalmefene (ng/mL) 0.250 1.93 1.00 2.94 2.00 4.06 4.00 4.69 8.00 4.86 24.0 5.47 48.0 5.43 72.0 5.46 144 3.87 312 3.87 480 4.60 648 5.29 816 3.16 984 2.32 1152 2.14 1320 1.45 1488 1.25 1656 1.05 1824 1.14 1992 1.09 2160 0.971 2328 0.798 2496 0.788 2664 0.719

Time vs nalmefene concentration data for compound 24 at 123 mg/kg is provided in Table 41.

TABLE 41 Time Mean conc Nalmefene (h) (ng/mL) 0.250 1.89 1.00 2.38 2.00 3.28 4.00 4.14 8.00 4.32 24.0 6.20 48.0 5.56 72.0 5.03 144 4.54 312 5.42 480 9.14 648 10.6 816 8.71 984 6.46 1152 6.57 1320 3.41 1488 2.64 1656 1.93 1824 1.29 1992 1.13 2160 0.821 2328 0.763 2496 0.457 2664 0.562

Time vs nalmefene concentration data for compound 24 at 165 mg/kg is provided in Table 42.

TABLE 42 Time Mean conc Nalmefene (h) (ng/mL) 0.250 3.48 1.00 4.90 2.00 5.23 4.00 6.41 8.00 7.24 24.0 8.44 48.0 8.24 72.0 7.80 144 9.93 312 12.8 480 18.0 648 16.6 816 15.3 984 9.14 1152 5.62 1320 5.44 1488 3.62 1656 3.90 1824 2.69 1992 1.81 2160 1.28 2328 1.34 2496 0.886 2664 0.591

Time vs naltrexone concentration data for compound 29 at 165 mg/kg (at 400 mg/ml in sesame oil) is provided in Table 43a.

TABLE 43a Time Mean conc Naltrexone (h) (ng/mL) 0.250 13.8 1.00 30.4 2.00 39.6 4.00 53.2 8.00 53.1 24.0 46.2 48.0 34.3 72.0 30.4 144 39.6 312 28.4 480 13.1 648 6.00 816 4.56 984 3.88 1152 3.71 1320 2.71 1488 2.70 1656 2.45 1824 2.25 1992 1.92

Time vs naltrexone concentration data for compound 29 (at 300 mg/ml in cottonseed oil, n=2) at 165 mg/kg is provided in Table 43b.

TABLE 43b Time Mean conc Naltrexone (h) (ng/mL) 0.250 29.0 1.00 40.1 2.00 69.3 4.00 91.4 8.00 90.5 24.0 92.1 48.0 66.9 72.0 54.7

Example 2: Dog pharmacokinetic studies

Purpose

The purpose of this study is to determine the pharmacokinetics of test compounds in plasma, following deep intramuscular administration to Beagle dogs (n=3, unless otherwise specified).

Acclimation/Quarantine

Animals are assessed as to their general health and acclimated for at least 5 days before being placed on study.

Animal Husbandry

Animals are pair housed during acclimation and individually housed during the study. The room(s) will be controlled and monitored for relative humidity (targeted mean range 40% to 70%, and any excursion from this range for more than 3 hours will be documented as a deviation) and temperature (targeted mean range 18° to 26° C., and any excursion from this range will be documented as a deviation) with 10 to 20 air changes/hour. The room will be on a 12-hour light/dark cycle except when interruptions are necessitated by study activities. Animals will be fed twice daily. Stock dogs will be fed approximately 220 grams of Certified Dog Diet daily (Beijing Keao Xieli Feed Co., Ltd. Beijing, P. R. China). These amounts can be adjusted as necessary based on food consumption of the group or an individual body weight changes of the group or an individual and/or changes in the certified diet. Reverse osmosis (RO) water is available to all animals, ad libitum. RO water is analyzed every three months and every batch of feed is analyzed before using. Enrichment toys are provided.

Animal Body Weights and Clinical Observation

Body weights were determined before selection to the study and on the day of dose administration. Weight monitoring was done every week.

Detailed clinical observation including behavior and activity, reflection, respiration, skin and fur, facial feature, genitourinary system, and other gross lesions was performed on the dosing day and at each sample collection time point.

Dose Administration

The dose formulation (concentration—400 mg base equivalents/ml in sesame oil+1% benzyl alcohol, unless otherwise specified) was administered via deep intramuscularly (unless otherwise specified). The injection vehicle was also dosed via deep intramuscular route (unless otherwise specified) on contralateral site of each animal at study initiation. The animals were sedated with Propofol at 6 mg/kg via IV administration. Following sedation hair was carefully removed from around the injection site and the area gently cleaned. Care will be taken to avoid irritating skin during shaving and cleaning the injection site. Then dogs will be dosed with deep IM administration. At least 2.5 cm depth from the surface into the central aspect of the quadriceps or biceps femoris muscle, by angling the needle toward the femur. If the needle hits the femur, simply draws back slightly and then inject. The dose volume will be determined by the animals' body weight collected on the morning of dosing day. For repeated administration, the injection sites may be rotated to minimize tissue injury.

Sample Collection

Blood samples were collected from a peripheral vessel from restrained, non-sedated animals per sampling time point.

Approximately 0.8 mL blood will be collected at each time point. All blood samples will be transferred into pre-chilled plastic microcentrifuge tubes containing 20 μL of 160 mg/mL sodium fluoride/potassium oxalate (NaF/KO=1/3) with 5% PMSF(100 mM in ethanol) as stabilizer and 16 μL of EDTA-K2 (0.5M) as anti-coagulant and placed on wet ice until centrifugation.

Each collected blood will be in the wet-ice before centrifuge.

Plasma Processing

Each collected blood sample was centrifuged for 4 minutes at 4° C. and 10000 rpm for plasma collection. Plasma was collected and transferred into a pre-labeled PP tube in dry ice at each time point and precipitated immediately using ACN at a ratio of 1:4 (plasma:ACN). Centrifuged again (10 minutes, 12000 rpm) and obtain the supernatant.

After terminal collection, all supernatant was stored at approximately −80° C. F until bioanalysis.

Bioanalytical Method and Sample Analysis

LC-MS/MS methods for the quantitative determination of test compound in biological matrix were developed. A calibration curve with 8 non-zero calibration standards were applied for the method including LLOQ (0.05 ng/ml). The sample analysis was performed concurrently with a set of calibration standards and two sets of QC samples using the LC-MS/MS method.

Data Analysis

Plasma concentration versus time data was analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. C_(max), T_(max), T_(1/2), AUC_((0-t)), AUC_((0-inf)), MRT_((0-t)),

MRT_((0-inf)) and graphs of plasma concentration versus time profile were prepared.

TABLE 44 Half-life Terminal Half- AUC_(0-inf) Nominal t_(1/2) life t_(1/2) (hour) AUC_(0-inf) (ng · h/mL) IM Dose (hour) nalmefene or (ng · h/mL) nalmefene or Compound (mg/kg) Vehicle prodrug naltrexone prodrug naltrexone Status 6 30 Sesame oil + 1% Benzyl 95 Data pending 398 6100 Ongoing Alcohol 48 Sesame oil + 1% Benzyl 135 277.9 1025 11464 Complete (shallow) Alcohol 48 Sesame oil + 1% Benzyl 54 552.6 2186 10619 Complete (deep) Alcohol 96 Sesame oil + 1% Benzyl 88 Data pending 1290 17090 Ongoing Alcohol 15 30 Sesame oil + 1% Benzyl 134 Data pending 521 682 Ongoing Alcohol 48 Sesame oil + 1% Benzyl 518 4244.8 1144 15021 Ongoing Alcohol 96 Sesame oil + 1% Benzyl 162 Data pending 1353 3789 Ongoing Alcohol 24 48 Cottonseed + 1% Benzyl 102 308.8 526 8500 Complete (shallow) Alcohol 48 Cottonseed + 1% Benzyl 59.6 404.0 613 7226 Complete (deep) Alcohol 45 30 Cottonseed + 1% Benzyl 151 Data pending 100 13069 Ongoing Alcohol 48 Cottonseed + 1% Benzyl 171 Data pending 267 10973 Ongoing Alcohol 96 Cottonseed oil + 1% None Data pending None 10215 Ongoing Benzyl Alcohol detected detected 7 48 Sesame oil + 1% Benzyl 201 Data pending 610 2145 Ongoing Alcohol 8 48 Sesame oil + 1% Benzyl 128 Data pending 264 8980 Ongoing Alcohol 1 48 Sesame oil + 1% Benzyl 261 Data pending 1040 3708 Ongoing Alcohol 3 48 Sesame oil + 1% Benzyl 63.1 Data pending 360 9084 Complete Alcohol 4 48 Sesame oil + 1% Benzyl 28 Data pending 829 6535 Complete Alcohol 5 48 Sesame oil + 1% Benzyl 70.9 Data pending 3869 8579 Complete Alcohol 35 48 Sesame oil + 1% Benzyl 70 Data pending None None Complete Alcohol detected detected Time vs nalmefene concentration data for compound 6 at 30 mg/kg is provided in Table 45.

TABLE 45* Time Mean conc Nalmefene (h) (ng/mL) 0.25 8.07 1 11.50 2 7.75 4 4.33 8 3.41 24 10.14 48 10.82 72 12.15 168 10.20 336 5.24 504 2.86 672 3.73 *study is ongoing Time vs nalmefene concentration data for compound 6 at 48 mg/kg (shallow IM injection) is provided in Table 46.

TABLE 46 Time Mean conc Nalmefene (h) (ng/mL) 0.25 4.75 1 5.03 2 3.98 4 5.26 8 5.40 34 10.25 48 14.50 72 21.40 196 26.80 336 15.37 504 8.34 672 5.15 840 2.18 1008 1.82 1176 1.53 1344 1.07 1512 0.53 Time vs nalmefene concentration data for compound 6 at 48 mg/kg (deep IM injection; redosed in dogs from Table 46) is provided in Table 47a.

TABLE 47a* Time Mean conc Nalmefene (h) (ng/mL) 0.25 8.99 1 18.97 2 13.23 4 9.70 8 11.08 24 12.91 48 15.83 72 19.93 168 21.93 336 12.00 504 5.59 672 4.31 840 2.24 1008 2.07 1176 1.86 1344 1.26 1512 1.11 2424 0.37 Time vs nalmefene concentration data for compound 6 at 48 mg/kg (deep IM injection; single dose in naïve dogs n=2) is provided in Table 47b.

TABLE 47b* Time Mean conc Nalmefene (h) (ng/mL) 0.25 17.3 1 17.0 2 13.2 4 9.99 8 7.73 24 12.3 48 17.6 72 25.0 168 13.7 336 6.87 504 5.26 672 2.96 *study is ongoing Time vs nalmefene concentration data for compound 6 at 96 mg/kg is provided in Table 48.

TABLE 48* Time Mean conc Nalmefene (h) (ng/mL) 0.25 13.59 1 24.17 2 14.11 4 12.56 8 6.84 24 17.80 48 22.13 72 33.97 168 41.9 336 22.2 504 13.3 672 7.18 *study is ongoing Time vs nalmefene concentration data for compound 15 at 30 mg/kg is provided in Table 49.

TABLE 49* Time Mean conc Nalmefene (h) (ng/mL) 0.25 5.74 1 10.04 2 5.49 4 2.84 8 0.67 24 0.41 48 0.56 72 0.78 168 0.578 336 0.437 504 0.385 672 0.401 *study is ongoing Time vs nalmefene concentration data for compound 15 at 48 mg/kg is provided in Table 50a.

TABLE 50a* Time Mean conc Nalmefene (h) (ng/mL) 0.25 6.74 1 15.67 2 10.28 4 4.23 8 1.19 24 1.65 48 1.95 72 3.14 168 2.92 336 3.94 504 2.71 672 1.96 840 1.90 1008 2.10 1176 1.70 1344 2.40 1512 2.44 2064 1.75 2232 1.58 2400 1.72 2568 1.46 2736 2.06 2904 2.19 3072 1.67 3240 1.32 3408 1.32 *study is ongoing Time vs nalmefene concentration data for compound 15 at 48 mg/kg (repeat of study from Table 50a) is provided in Table 50b.

TABLE 50b* Time Mean conc Nalmefene (h) (ng/mL) 0.25 7.55 1 12.3 2 10.3 4 4.48 8 1.55 24 0.965 48 1.53 72 1.64 168 2.32 336 2.08 504 1.28 672 1.47 *study is ongoing Time vs nalmefene concentration data for compound 15 at 96 mg/kg is provided in Table 51.

TABLE 51* Time Mean conc Nalmefene (h) (ng/mL) 0.25 18.63 1 24.13 2 16.83 4 9.81 8 2.84 24 2.38 48 2.65 72 2.98 168 3.61 336 4.06 504 3.03 672 2.32 *study is ongoing Time vs nalmefene concentration data for compound 18 at 48 mg/kg is provided in Table 52.

TABLE 52 Time Mean conc Nalmefene (h) (ng/mL) 0.25 3.49 1 5.44 2 5.18 4 6.62 8 9.77 24 14.40 48 14.24 72 16.13 Time vs nalmefene concentration data for compound 24 at 48 mg/kg (shallow IM injection) is provided in Table 53.

TABLE 53 Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.33 1 1.62 2 2.17 4 4.70 8 4.71 24 8.34 48 10.93 72 15.83 168 24.07 336 11.29 504 5.13 672 2.42 840 1.59 1008 1.26 1176 0.86 1344 0.56 1512 0.50 Time vs nalmefene concentration data for compound 24 at 48 mg/kg (deep IM injection) is provided in Table 54.

TABLE 54 Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.64 1 2.03 2 2.11 4 2.23 8 3.47 24 5.82 48 11.60 72 15.90 168 14.63 336 9.96 504 5.85 672 2.47 840 1.99 1008 1.51 1176 1.07 1344 0.85 1512 0.54 Time vs nalmefene concentration data for compound 45 at 30 mg/kg is provided in Table 55.

TABLE 55* Time Mean conc Nalmefene (h) (ng/mL) 0.25 2.82 1 4.04 2 3.24 4 2.75 8 2.61 24 3.00 48 5.31 72 4.72 168 6.26 336 4.36 504 3.09 672 3.51 *study is ongoing

TABLE 56* Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.22 1 2.23 2 1.77 4 1.75 8 1.95 24 3.05 48 10.00 72 10.05 168 18.87 336 14.83 504 7.43 672 3.24 840 4.06 1008 2.49 1176 2.17 1344 2.16 1512 2.18 1680 2.00 1848 1.39 2016 1.33 2184 1.06 *study is ongoing Time vs nalmefene concentration data for compound 45 at 48 mg/kg is provided in Table 56.

TABLE 57* Time Mean conc Nalmefene (h) (ng/mL) 0.25 2.93 1 7.71 2 6.64 4 4.86 8 3.66 24 7.14 48 11.30 72 9.32 168 17.8 336 16.5 504 6.81 672 5.30 *study is ongoing Time vs naltrexone concentration data for compound 7 at 24 mg/kg (n=2) is provided in Table 58.

TABLE 58* Time Mean conc Naltrexone (h) (ng/mL) 0.25 3.61 1 5.25 2 2.52 4 0.82 8 0.29 24 0.61 48 0.61 72 0.67 168 1.75 336 2.29 504 2.44 672 2.19 *study is ongoing Time vs naltrexone concentration data for compound 7 at 48 mg/kg is provided in Table 59.

TABLE 59* Time Mean conc Naltrexone (h) (ng/mL) 0.25 18.23 1 18.13 2 9.67 4 3.35 8 1.33 24 1.43 48 2.30 72 2.41 168 1.85 336 1.09 504 1.35 672 1.27 840 1.95 1008 1.54 1176 1.02 1344 1.03 1512 1.06 *study is ongoing Time vs naltrexone concentration data for compound 8 at 48 mg/kg is provided in Table 60.

TABLE 60* Time Mean conc Naltrexone (h) (ng/mL) 0.25 103.20 1 159.67 2 52.33 4 9.74 8 5.03 24 3.46 48 4.06 72 4.87 168 7.28 336 3.91 504 9.72 672 4.59 840 2.52 1008 2.70 1176 1.71 1344 1.63 1512 1.60 *study is ongoing Time vs nalmefene concentration data for compound 1 at 48 mg/kg is provided in Table 61.

TABLE 61* Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.90 1 4.24 2 2.26 4 0.92 8 0.57 24 0.87 48 1.41 72 1.84 168 2.57 336 1.78 504 1.49 672 1.21 840 1.64 1008 1.85 1176 1.58 1344 1.24 1512 1.19 *study is ongoing Time vs nalmefene concentration data for compound 3 at 48 mg/kg is provided in Table 62.

TABLE 62 Time Mean conc Nalmefene (h) (ng/mL) 0.25 2.54 1 4.48 2 5.37 4 9.26 8 13.37 24 25.43 48 37.57 72 62.60 168 27.77 336 4.50 504 0.97 672 0.34 Time vs naltrexone concentration data for compound 4 at 48 mg/kg is provided in Table 63.

TABLE 63 Time Mean conc Naltrexone (h) (ng/mL) 0.25 21.90 1 38.67 2 41.17 4 74.47 8 78.07 24 53.23 48 52.93 72 70.03 168 3.76 336  ND** 504 ND 672 ND **ND = none detected Time vs naltrexone concentration data for compound 5 at 48 mg/kg (n=2) is provided in Table 64.

TABLE 64 Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.12 1 2.75 2 3.55 4 5.49 8 6.67 24 17.40 48 26.95 72 30.65 168 27.85 336 11.00 504 2.07 672 1.06 Time vs naltrexone concentration data for compound 35 at 48 mg/kg is provided in Table 65.

TABLE 65 Time Mean conc Nalmefene (h) (ng/mL) 0.25 9.57 1 19.07 2 14.50 4 18.80 8 25.80 24 103.87 48 174.67 72 237.00

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA Day 0 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 1 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 2 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 3 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 4 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 5 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 6 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 7 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 14 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 21 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 28 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 35 No obvious No obvious clinical No obvious No obvious No obvious No obvious clinical observation clinical clinical observation clinical clinical observation observation observation observation Day 39 No obvious No obvious clinical a few erythema regression of erythema and No obvious No obvious clinical observation on the inside escharosis on the outside clinical clinical observation of the left of the hindleg/a few erythema observation observation foreleg on the inside of the right foreleg Day 41 No obvious No obvious clinical a few new slight rashes and escharosis No obvious No obvious clinical observation erythema on on the outside of the right leg/ clinical clinical observation the inside of a few erythema on the observation observation the left foreleg inside of the right foreleg Day 42 slight rashes slight rashes on the outside a few erythema new slight rashes and escharosis No obvious No obvious on the left of right thigh/ on the inside on the outside of the right leg/ clinical clinical thigh and crus slight rashes on of the a few erythema on observation observation the right crus left foreleg the inside of the right foreleg Day 43 slight rashes slight rashes on the outside a few erythema new slight rashes and escharosis No obvious No obvious on the left of right thigh/slight rashes on the inside on the outside of the right leg/ clinical clinical thigh and crus on the right crus of the left a few erythema on the inside of observation observation foreleg the right foreleg Day 46 slight rashes and slight rashes and escharosis a few erythema a few erythema on the No obvious No obvious escharosis on the outside of right on the inside of outside of the thigh clinical clinical on the left thigh/slight rashes on the the left foreleg, observation observation thigh and crus right crus fresh erythema on the outside of the thigh Day 48 slight rashes and slight rashes and escharosis slight rashes slight rashes and escharosis No obvious No obvious escharosis of slight on the outside of right on the inside on the outside clinical clinical rashes on the thigh/slight rashes on the of the left of the thigh/slight observation observation outside of left right crus foreleg/slight rashes on the inside of foreleg leg/slight rashes rashes on the on the inside outside of and outside the left leg of the crus Day 49 slight rashes and slight rashes and slight rashes slight rashes and escharosis No obvious No obvious escharosis of slight escharosis on the inside of on the outside clinical clinical rashes on the outside on the outside the left foreleg/ of the thigh/slight rashes observation observation of left leg/slight of right thigh/ slight rashes on the inside of foreleg rashes on the inside slight rashes on the outside and outside on the right crus of the left leg of the crus Day 53 slight rashes and Recovery of rashes on No obvious slight rashes on the No obvious No obvious escharosis of slight the outside of the leg clinical outside of the leg/ clinical clinical rashes on the outside observation slighl rashes on the inside observation observation of left leg/slight of foreleg rashes on the inside and outside of the crus Day 55 escharosis of slight No obvious clinical No obvious slight rashes on the No obvious No obvious rashes on the observation clinical outside of the leg/several clinical clinical outside of left leg/ observation rashes on the inside of foreleg observation observation several rashes on the outside of the crus Day 56 No obvious No obvious clinical No obvious escharosis of rashes on No obvious No obvious clinical observation clinical the outside of the leg/ clinical clinical observation observation no obvious clinical observation observation observation on the inside of foreleg Day 60 No obvious No obvious clinical No obvious escharosis of rashes on No obvious No obvious clinical observation clinical the outside of the leg/ clinical clinical observation observation no obvious clinical observation observation observation on the inside of foreleg

time D1001 D1002 D1003 point R-TA L-vehicle R-TA L-vehicle R-TA L-vehicle 8 hr No obvious NA No obvious NA No obvious NA clinical observation clinical observation clinical observation 72 hr No obvious NA No obvious NA Slight swelling NA clinical observation clinical observation Day 5 No obvious NA Slight swelling NA Larger swelling NA clinical observation Day 7 No obvious NA Slight swelling NA Larger swelling NA clinical observation Day 8 Slight swelling, NA Slight swelling, NA Larger swelling, NA induration induration induration Day 9 The same with NA The same with Day 8 NA The same with Day 8 NA Day 8 Day 12 Not obvious swelling NA Not obvious swelling NA Obvious swelling and NA and induration, no and induration, no induration, no inflammation, painless inflammation, painless inflammation, painless with touching with touching with touching Day 16 Same with Day 15 No obvious Same with Day 15 No obvious Same with Day 15 No obvious clinical clinical clinical observation observation observation Day 17 More indurated than No obvious More indurated No obvious More indurated than No obvious the vehicle site clinical observation than the vehicle site clinical the vehicle site clinical observation observation Day 18 The same with No obvious The same with No obvious The same with No obvious Day 17 clinical observation Day 17 clinical Day 17 clinical observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation Day 22 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation Day 30 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation Day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation Day 42 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation Day 49 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation Day 56 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation Day 63 No obvious No obvious No obvious No obvious No obvious No obvious clinical observation clinical observation clinical observation clinical clinical observation clinical observation observation

time D1001 D1002 D1003 point L-TA R-vehicle L-TA R-vehicle L-TA R-vehicle 8 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 1 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 4 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 5 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 6 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 14 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 28 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 42 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 49 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 56 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 63 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 101 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation

Clinical Observations for Dogs Treated with Compound 6 at 48 mg/kg (Deep IM Injections, Single Dose in Naïve Dogs n=2) are Provided in Table 68b

time D1001 D1002 point L-vehicle R-TA L-vehicle R-TA Day 0 No obvious No obvious vomit about 20 g chyme clinical clinical at 1 hr post dose observation observation Day 3 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 7 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 14 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 21 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 28 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 35 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 42 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation

Clinical Observations for Dogs Treated with Compound 6 at 96 mg/kg are Provided in Table 69

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA Day 0 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 1 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 4 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 5 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 6 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 14 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 42 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 49 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation Day 56 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical observation clinical observation clinical observation clinical observation clinical observation observation

Clinical Observations for Dogs Treated with Compound 15 at 30 mg/kg are Provided in Table 70

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA Day 0 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 1 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 2 No obvious No obvious No obvious No obvious No obviou No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 4 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 5 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 6 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical observation clinical observation clinical clinical observation observation observation observation Day 14 No obvious No obvious large area of red spots large area of red spots on the No obvious No obvious clinical clinical on the outside and outside and inside of the leg/ clinical clinical observation observation inside of the leg/the the red spots of outsidewere observation observation red spots of outside in decrustation were in decrustation Day 18 No obvious No obvious large area of red large area of red spots No obvious No obvious clinical clinical spots on the outside on the outside of the clinical clinical observation observation of the leg/the red leg/the red spots of outside observation observation spots of outside in decrustation were in decrustation and escharosis and escharosis Day 20 No obvious No obvious regression of red regression of red spots No obvious No obvious clinical clinical spots on the outside on the outside of the clinical clinical observation observation of the leg and leg escharosis/scap 1.5 cm * observation observation decrustation 1.5 cm on the inside of right leg Day 21 No obvious No obvious decrustation on the escharosis on the the No obvious No obvious clinical clinical outside of the left leg outside of the leg/ clinical clinical observation observation scap 1.5 cm * 1.5 cm on observation observation inside of right leg Day 25 No obvious No obvious decrustation on the decrustation on the No obvious No obvious clinical clinical outside of the left leg outside of the right leg clinical clinical observation observation observation observation Day 27 No obvious No obvious escharosis and erythema, escharosis No obvious No obvious clinical clinical decrustation on the outside and decrustation on the clinical clinical observation observation of the left leg/erythema outside of the right leg/ observation observation on the inside of the left leg erythema on the inside of the right leg Day 28 No obvious No obvious decrustation on the decrustation on the No obvious No ohvious clinical clinical outside of the left leg outside of the right leg clinical clinical observation observation observation observation Day 32 No obvious No obvious escharosis on the outside escharosis on the No obvious No obvious clinical clinical of the left leg/erythema outside of the right clinical clinical observation observation on the inside of the left leg leg/erythema on the observation observation inside of the right leg Day 34 No obvious No obvious escharosis on the outside of escharosis and a few No obvious No obvious clinical clinical the left leg/erythema erythema on the clinical clinical observation observation on the inside of the left leg outside of the right observation observation leg/erythema on the inside of the right leg Day 35 No obvious No obvious escharosis on the outside escharosis and a few No obvious No obvious clinical clinical of the left leg/erythema on erythema on the clinical clinical observation observation the inside of the left leg outside of the right observation observation leg/erythema on the inside of the right leg Day 39 No obvious No obvious recovery for escharosis/ recovery for escharosis/several No obvious No obvious clinical clinical several rashes and slight rashes and slight decrustation clinical clinical observation observation decrustation on the on the outside of the observation observation outside of the left right leg/several rashes leg/ several rashes on the inside of the on the inside of the left leg left leg Day 41 No obvious No obvious recovery for escharosis/several recovery for escharosis/ No obvious No obvious clinical clinical rashes and slight decrustation several rashes and clinical clinical observation observation on the outside of the left slight decrustation on the observation observation leg/several rashes on the outside of the right leg/several inside of the left leg rashes on the inside of the left leg Day 42 No obvious No obvious recovery for escharosis/several recovery for escharosis/several No obvious No obvious clinical clinical rashes and slight rashes and slight decrustation clinical clinical observation observation decrustation on the outside of on the outside of the observation observation the left leg/several rashes on right leg/several rashes the inside of the left leg on the inside of the left leg Day 43 No obvious No obvious recovery for escharosis/several recovery for escharosis/ No obvious No obvious clinical clinical rashes and slight decrustation several rashes and slight clinical clinical observation observation on the outside of the left decrustation on the outside observation observation leg/several rashes of the right leg/several rashes inside of the left leg on on the inside of the left leg Day 46 No obvious No obvious the recovery for escharosis/ recovery for escharosis/several No obvious No obvious clinical clinical several rashes and slight rashes and slight decrustation clinical clinical observation observation decrustation on the outside on the outside of the observation observation of the left leg/several rashes right leg/several rashes on the inside of the left leg on the inside of the left leg Day 49 No obvious No obvious recovery for escharosis/ recovery for escharosis/ No obvious No obvious clinical clinical several rashes and slight several rashes and clinical clinical observation observation decrustation on the outside slight decrustation on the observation observation of the left leg/several rashes outside of the right leg/several on the inside of the left leg rashes on the inside of the left leg Day 53 No obvious No obvious Recovery for rashes on Recovery for rashes No obvious No obvious clinical clinical the leg on the leg clinical clinical observation observation observation observation Day 56 No obvious No obvious No obvious No obvious clinical No obvious No obvious clinical clinical clinical observation observation clinical clinical observation observation observation observation Day 59 No obvious No obvious No obvious No obvious clinical No obvious No obvious clinical clinical clinical observation observation clinical clinical observation observation observation observation

time D1501 D1502 D1503 point L-TA R-vehicle L-TA R-vehicle L-TA R-vehicle before No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical day 7 observation observation observation observation observation observation day 7 No obvious clinical No obvious clinical Swelling for the No obvious clinical No obvious clinical No obvious clinical observation observation whole leg observation observation observation day 8 No obvious clinical No obvious clinical Swelling at upper No obvious clinical No obvious clinical No obvious clinical observation observation leg/edema at lower observation observation observation leg day 9 No obvious clinical No obvious clinical Swelling at upper No obvious clinical No obvious clinical No obvious clinical observation observation leg/edema at lower observation observation observation leg/skin rashes at groin day 10 No obvious clinical No obvious clinical Swelling at the No obvious clinical No obvious clinical No obvious clinical observation observation upper leg/skin observation observation observation rashes at groin day 11 No obvious clinical No obvious clinical Swelling 9 * 7 cm at No obvious clinical No obvious clinical No obvious clinical observation observation the upper leg/skin observation observation observation rashes at groin day 12 No obvious clinical No obvious clinical Swelling 9 * 7 cm at No obvious clinical No obvious clinical No obvious clinical observation observation the upper leg/skin observation observation observation rashes at groin day 13 No obvious clinical No obvious clinical Slight swelling at No obvious clinical No obvious clinical No obvious clinical observation observation upper leg/skin observation observation observation rashes at groin day 14 No obvious clinical No obvious clinical Slight swelling at No obvious clinical No obvious clinical No obvious clinical observation observation upper leg/skin observation observation observation rashes at groin day 15 No obvious clinical No obvious clinical Slight swelling at No obvious clinical No obvious clinical No obvious clinical observation observation upper leg/skin observation observation observation rashes at groin day 16 No obvious clinical No obvious clinical Slight swelling at No obvious clinical No obvious clinical No obvious clinical observation observation upper leg/skin observation observation observation rashes at groin day 17 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 21 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 28 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 35 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 42 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 49 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 56 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 63 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 86 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day 93 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 100 observation observation observation observation observation observation day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 107 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 114 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 121 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 128 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 135 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 142 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 149 observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 15 at 48 mg/kg are Provided in Table 71b

time D1001 D1002 D1003 point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 0 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 3 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 7 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 14 observation observation observation observation observation observation Day No obvious clinical several rashes on the several rashes on the several rashes on the several rash on the several rash on the 21 observation outside of the right outside of the left leg outside of the right leg outside of the left outside of the right leg leg leg Day a few rashes of the a few rashes on the lots of rashes on the escharosis of the rashes a few rashes of the a few rashes and 24 left leg outside of the right outside of the left leg part on the outside of the left leg decrustation on the leg right leg outside of the right leg Day escharosis of the Fading in rashes and lots of rashes on the lots of rashes on the several rashes on several rashes and slight 26 several rashes part escharosis on the outside of the left outside of the right the outside of the decrustation on the on the outside of outside of the right leg/escharosis of the leg/escharosis of half left leg/escharosis outside of the right leg the left leg leg half rashes rashes of rashes part Day escharosis of the Fading in rashes and lots of rashes on the lots of rashes on the several rashes on several rashes and slight 28 several rashes part escharosis on the outside of the left outside of the right the outside of the decrustation on the on the outside of outside of the right leg/escharosis of half leg/escharosis of half left leg/escharosis outside of the right leg the left leg leg/new several rashes rashes of rashes rashes on the right crus Day escharosis of the Fading in rashes and escharosis of the escharosis and rashes on several rashes on several rashes and slight 31 several rashes part escharosis on the several rashes part on the outside of the leg the outside of the decrustation on the on the outside of outside of the right the outside of the left left leg/escharosis outside of the right leg the left leg leg/new several leg of rashes rashes on the right crus Day escharosis of the Fading in several recovery for rashes recovery for rashes on several rashes on several rashes and slight 33 several rashes on rashes and escharosis on the outside of the the outside of the right the outside of the decrustation on the the left leg on the outside of the left leg/only a few leg/only a few rash, left leg/escharosis outside of leg right leg/new several rash, escharosis and escharosis and of rashes rashes on the right decrustation decrustation crus Day rashes subside on rashes subside on the recovery for rashes rashes subside on the leg several rashes and several rashes and slight 35 the leg outside of leg/several on the outside of the escharosis on the decrustation on the rashes on the right left leg/only a few outside of the left outside of leg crus rash, escharosis and leg decrustation Day No obvious clinical No obvious clinical No obvious clinical Fading in rashes on the No obvious clinical No obvious clinical 38 observation observation observation leg observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 40 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 42 observation observation observation observation observation observation Day No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical 45 observation observation observation observation observation observation

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA Day 0 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 1 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 2 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 3 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 4 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 5 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 6 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 7 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical Swelling 8 * 4 cm observation observation observation observation observation Day 14 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 21 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 28 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 35 No obvious clinical Slight rashes on the No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation outside of the observation observation observation observation hindlimb Day 38 Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the outside of the outside of the outside of the upper outside of the upper outside of the outside of the hindlimb hindlimb hindlimb hindlimb hindlimb, skin hindlimb, skin exuviating exuviating Day 40 Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the outside of the outside of the upper outside of the upper outside of the upper outside of the outside of the hindlimb hindlimb were hindlimb were hindlimb hindlimb, skin hindlimb, skin disappearing, new disappearing, new exuviating exuviating rashes appeared on rashes appeared on the inside. the inside. Day 42 New rashes appeared No obvious clinical Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the on the inside of the observation inside of the inside of the hindlimb outside of the outside of the hindlimb hindlimb hindlimb hindlimb Day 45 Slight rashes New rashes appeared Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the appeared on the on the inside of the inside of the inside of the hindlimb outside of the outside of the inside of the hindlimb hindlimb hindlimb, skin hindlimb, some hindlimb exuviating were scabbing Day 47 Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the inside of the inside of the inside of the inside of the hindlimb outside of the outside of the hindlimb hindlimb hindlimb hindlimb, skin hindlimb, some exuviating were scabbing Day 49 Slight rashes Slight rashes Slight rashes on the Slight rashes on the Slight rashes on the Slight rashes on the scabbing on the scabbing on the inside of the inside of the hindlimb outside of the outside of the inside of the inside of the hindlimb hindlimb, skin hindlimb, some hindlimb hindlimb exuviating were scabbing Day 52 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 54 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation Day 56 No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical No obvious clinical observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 18 at 48 mg/kg are Provided in Table 73

time D1501 D1502 point L-vehicle R-TA L-vehicle R-TA  8 hr No obvious No obvious clinical No obvious No obvious clinical clinical observation clinical observation observation observation  24 hr No obvious Induration at the upper Red spots at the Swelling 15 * 12 cm at the clinical leg 18 * 10 cm/swelling at upper leg upper leg/swelling 8 * 11 at observation the inner upper leg inner upper leg with redness 6 * 9 cm/BT: 39.1° C. and warmth/swelling and deep redness at the calf/BT: 40.1° C.  48 hr swelling Induration at the upper Red spots at the Swelling 15 * 12 cm at the 3 * 6 cm leg 18 * 10 cm/swelling at upper leg upper leg/swelling 8 * 11 at the inner upper leg inner upper leg with redness 8 * 9 cm/Red spots at the 1 and warmth/swelling and 'clock black at the calf/BT: 40.0° C. position/BT: 39.1° C.  72 hr swelling Induration at the upper Red spots at the Swelling 15 * 12 cm at the 3 * 6 cm leg 18 * 10 cm/swelling at upper leg upper leg/swelling 8 * 11 at the inner upper leg inner upper leg with redness 8 * 9 cm/Red spots at the 1 and warmth/swelling and 'clock black at the position/BT: 39.1° C./thin/inappetence calf/BT: 38.7° C./ulcerated scab 2 * 4 at inner upper leg/thin/inappetence  96 hr swelling lame/serious induration slight lame/induration of the whole 3 * 6 cm of the whole leg/edema swelling/red leg/edema at distal lower at ankle/Red spots at the spots at the leg/induration and black at 1 'clock upper leg proximal lower position//BT: 39.1° C./thin/ leg/necrosis(black and muscle inappetence/weight atrophy) 1.5 * 1.5 cm at upper decreases from 6.71 kg to leg and 6 * 2 cm at lower leg 6.0 kg/bloody stools with red secreta/BT: 39.0° C./thin/inappetence/ weight decreases from 7.76 kg to 6.86 kg/bloody stools 120 hr swelling lame/serious induration slight lame/induration of the whole 3 * 6 cm of the whole leg/edema swelling/red leg/edema at distal lower at ankle/Red spots at the spots at the leg/induration and black at 1 'clock upper leg proximal lower position//BT: 39.8° C./thin/ leg/necrosis(black and muscle inappetence atrophy) 1.5 * 1.5 cm at upper leg and 6 * 2 cm at lower leg with red secreta/BT: 39.1° C./thin/inappetence 144 hr swelling lame/serious induration slight lame/induration of the whole 3 * 6 cm of the whole leg/edema swelling/red leg/edema at distal lower at ankle/Red spots at the spots at the leg/induration and black at 1 'clock upper leg proximal lower position/induration at the leg/necrosis(black and muscle inner upper leg with atrophy) 1.5 * 1.5 cm at upper deep color and leg and 6 * 2 cm at lower leg warmth/BT: 39.3° C./thin/inappetence/ with red 6.18 kg secreta/BT: 39.2° C./thin/inappetence/ 6.92 kg 168 hr swelling lame/serious induration slight lame/induration of the whole 3 * 6 cm of the whole leg/edema swelling/red leg/edema at distal lower at ankle/Red spots at the spots at the leg/induration and black at 1 'clock upper leg proximal lower position/induration at the leg/necrosis(black and muscle inner upper leg with atrophy) 1.5 * 1.5 cm at upper deep color and leg and 6 * 2 cm at lower leg warmth/BT: 39.2° C./thin/inappetence/ with red 6.13 kg secreta/BT: 39.6° C./thin/inappetence/ 7.01 kg day 8  swelling lame/serious induration slight lame/induration of the whole 3 * 6 cm/ of the whole leg/edema swelling/red leg/edema at distal lower at ankle/ulceration with spots at the leg/induration and black at red and thick secreta at upper leg proximal lower upper leg/induration at leg/necrosis(black and muscle the inner upper leg with atrophy) 3 * 1.5 cm at upper leg deep color and and 6 * 4 cm at lower leg with warmth/BT: 39.4° C./thin/inappetence/ red secreta and muscle 6.11 kg exposure/ BT: 39.5° C./thin/inappetence/6.76 kg day 9  swelling lame/serious induration slight lame/induration of the whole 3 * 6 cm/ of the whole leg/edema swelling/red leg/edema at distal lower at ankle/3 parts of spots at the leg/induration and black at ulceration with yellow upper leg proximal lower and thick secreta at leg/necrosis(black and muscle upper leg/induration at atrophy) 3 * 1.5 cm at upper leg the inner upper leg with and 6 * 4 cm at lower leg with deep color and yellow secreta and muscle warmth/BT: 38.7° C./thin/inappetence/ exposure/ 6.09 kg BT: 38.5° C./thin/inappetence/6.79 kg day 10 swelling lame/serious induration No obvious induration of the whole 3 * 6 cm/ of the whole leg/edema clinical leg/edema at distal lower at ankle/3 parts of observation leg/induration and black at ulceration with yellow proximal lower and thick secreta at leg/necrosis(black and muscle upper leg one part of atrophy) 3 * 1.5 cm at upper leg ulceration with pink and and 6 * 4 cm at lower leg with thick secreta at inner yellow secreta and muscle upper leg/induration at exposure/ the inner upper leg with BT: 38.8° C./thin/inappetence/6.68 kg deep color and warmth/BT: 38.3° C./thin/inappetence/ 6.05 kg day 11 No obvious lame/serious induration No obvious induration of the whole clinical of the whole leg/edema clinical leg/edema at distal lower observation at ankle/3 parts of observation leg/induration and black at ulceration with yellow proximal lower and thick secreta at leg/necrosis(black and muscle upper leg one part of atrophy) 3 * 1.5 cm at upper leg ulceration with pink and and 6 * 4 cm at lower leg with thick secreta at inner yellow secreta and muscle upper leg/induration at exposure/ the inner upper leg with BT: 38.9° C./thin/inappetence/6.83 kg deep color and warmth/BT: 38.5° C./thin/inappetence/ 5.99 kg day 12 No obvious lame/serious induration No obvious induration of the whole clinical of the whole leg/edema clinical leg/edema at distal lower observation at ankle/3 parts of observation leg/induration and black at ulceration with yellow proximal lower and thick secreta at leg/necrosis(black and muscle upper leg one part of atrophy) 3 * 1.5 cm at upper leg ulceration at inner upper and 6 * 4 cm at lower leg with leg/induration at the yellow secreta and muscle inner upper leg with exposure/thin/inappetence/ deep color and warmth/thin/inappetence day 13 No obvious lame/swelling at the No obvious induration of the whole clinical inner upper leg with clinical leg/necrosis(black and muscle observation purple color/edema at observation atrophy) 1.5 * 1.5 cm at upper ankle//3 parts of leg and 5 * 1.5 cm at lower leg ulceration with yellow with muscle exposure/6.92 kg and thick secreta at upper leg one part of ulceration with pink and thick secreta at inner upper leg/6.06 kg day 14 No obvious lame/swelling at the No obvious induration of the whole clinical inner upper leg with clinical leg/necrosis(black and muscle observation purple color/edema at observation atrophy) 1.5 * 1.5 cm at upper ankle//3 parts of leg and 5 * 1.5 cm at lower leg ulceration with yellow with muscle exposure/6.93 kg and thick secreta at upper leg one part of ulceration with pink and thick secreta at inner upper leg/6.01 kg day 15 No obvious lame/swelling at the No obvious induration of the whole clinical inner upper leg with clinical leg/necrosis(black and muscle observation purple color/edema at observation atrophy) 1.5 * 1.5 cm at upper ankle//3 parts of leg and 5 * 1.5 cm at lower leg ulceration with yellow with muscle exposure and thick secreta at upper leg one part of ulceration with pink and thick secreta at inner upper leg time D1502 point L-vehicle R-TA  8 hr About 10 mL loose feces at 8 hr post dose  24 hr No obvious clinical Induration 15 * 13 cm for observation the whole leg with redness and warmth/Induration at the inner upper leg 9 * 11 cm with redness and warmth/BT: 39.9° C.  48 hr No obvious clinical Induration 15 * 13 cm for observation the whole leg with redness and warmth/Induration at the inner upper leg 9 * 11 cm with redness and warmth/BT: 39.9° C.  72 hr No obvious clinical Induration 15 * 13 cm for observation the whole leg with redness and warmth/Induration at the inner upper leg 9 * 11 cm with redness and warmth/BT: 39.4° C./thin/inappetence  96 hr No obvious clinical Induration for the whole observation leg with redness and warmth/BT: 39.4° C./thin/inappetence/ weight decreases from 6.52 kg to 6.17 kg/bloody stools 120 hr No obvious clinical Induration for the whole observation leg with redness and warmth/BT: 39.5° C./thin 144 hr No obvious clinical Induration for the whole observation leg with redness and warmth/BT: 39.5° C./thin/6.15 kg 168 hr No obvious clinical Induration for the whole observation leg with redness and warmth/ulceration at upper leg with 0.5 * 0.5 cm/BT: 38.7° C./thin/ 5.93 kg day 8  No obvious clinical Induration for the whole observation leg with redness and warmth/ulceration at upper leg with 0.5 * 0.5 cm and redness and warmth/Induration at the front lower leg and fluctuation with red and thick liquid at the back lower legBT: 38.7° C./thin/5.98 kg day 9  No obvious clinical Induration for the whole observation leg with redness and warmth/ulceration at upper leg with 0.5 * 0.5 cm and redness and warmth/Induration at the front lower leg and fluctuation with red and thick liquid at the back lower legBT: 38.8° C./thin/6.15 kg day 10 No obvious clinical Induration for the whole observation leg with redness and warmth/ulceration at upper leg with 0.5 * 0.5 cm and redness and warmth and becoming scab/Induration at the front lower leg and fluctuation with red and thick liquid at the back lower legBT: 38.7° C./thin/6.13 kg day 11 No obvious clinical Induration for the whole observation leg with redness and warmth/ulceration at upper leg with 0.5 * 0.5 cm and redness and warmth and becoming scab/Induration at the front lower leg and fluctuation with red and thick liquid at the back lower legBT: 38.7° C./thin/6.13 kg day 12 No obvious clinical Induration for the whole observation leg with redness and warmth/ulceration at upper leg with 0.5 * 0.5 cm and redness and warmth and becoming scab/thin day 13 No obvious clinical Induration for the whole observation leg/ulceration at upper leg with scab/6.45 kg day 14 No obvious clinical Induration for the whole observation leg/ulceration at upper leg with scab/6.38 kg day 15 No obvious clinical Induration for the whole observation leg/ulceration at upper leg with scab

D1001 D1002 time point L-vehicle R-TA L-vehicle  2 hr No obvious clinical Swelling No obvious clinical observation observation  8 hr No obvious clinical Larger swelling No obvious clinical observation observation  24 hr No obvious clinical Larger swelling No obvious clinical observation observation  48 hr No obvious clinical Larger swelling No obvious clinical observation observation  72 hr No obvious clinical Larger swelling No obvious clinical observation observation  96 hr No obvious clinical Larger swelling No obvious clinical observation observation 120 hr No obvious clinical Larger swelling with No obvious clinical observation induration observation 144 hr 0.1 * 0.2 cm/few red Larger swelling with 2 yellow scab with site induration 0.8 * 0.5 cm of each, one was yellow with red edge 178 hr 4 yellow scabs with induration/slight red/5 * 8 cm 3 yellow scab with 0.1 * 0.2 cm wide/0.5-0.8 cm depth 0.2 * 0.2 cm each/superficial each/superficial(one scab cover two red site) 192 hr 4 yellow scabs with induration/slight red/5 * 8 cm 2 yellow scabs with 0.1 * 0.2 cm wide/0.5-0.8 cm depth 0.2 * 0.2 each; 2 yellow each/superficial. scabs with 0.3 * 0.5 cm each 216 hr 4 yellow scabs with induration/slight red/4 * 8 cm Yellow 0.1 * 0.2 cm wide/0.5-0.8 cm depth scab/superficial/slight/shrinking each/superficial/ leasion shrinking 240 hr 4 yellow scabs with induration/7 * 7 cm wide Yellow 0.1 * 0.2 cm scab/superficial/slight/shrinking each/superficial/ leasion shrinking 264 hr 4 yellow scabs with Induration/7 * 7 cm wide/ Yellow 0.1 * 0.1 cm/ scab/superficial/slight/shrinking each/superficial/shrinking 288 hr Yellow Induration/7 * 7 cm wide/ The yellow scab/superficial/slight/ scab/disappeared, skin shrinking to repairing; a red spot 312 hr Yellow Induration/slight red/ skin repairing/the skin scab/superficial/slight/ 10 * 6 cm(longest * shortest, short of scab site; shrinking; boot shape) wide/0.5-0.8 cm New yellow 1 red depth; Skin rashes at scab/superficial/1.5 * 2 cm rash/0.2 * 0.2 cm groin and oxter/moderate 336 hr No obvious clinical Induration/slight red/ yellow observation 10 * 6 cm(longest * shortest, short scab/superficial/2 * 2 cm boot shape) wide/Skin rashes at groin 360 hr No obvious clinical Induration/slight red/ yellow observation 10 * 6 cm(longest * shortest, short scab/superficial/2 * 2 cm/ boot shape) wide/Skin alopecia rashes at groin and oxter 384 hr No obvious clinical Induration/slight red/ yellow observation 9.5 * 6 cm(longest * shortest, short scab/superficial/2 * 2 cm/ boot shape) wide/Skin alopecia rashes at groin and oxter/moderate 408 hr No obvious clinical Induration/slight red/ yellow observation 10 * 7 cm(longest * shortest, short scab/superficial/2 * 2 cm/ boot shape) wide/Skin alopecia rashes at groin 432 hr No obvious clinical Induration/slight red/ yellow observation 10 * 6 cm(longest * shortest, short scab/superficial/2 * 2 cm/ boot shape) wide/Skin alopecia rashes at groin and oxter 456 hr No obvious clinical Induration/slight red/ yellow observation 10 * 6 cm(longest * shortest, short scab/superficial/2 * 2 cm/ boot shape) wide/Skin alopecia rashes at groin and oxter 480 hr No obvious clinical Induration/slight red/ yellow observation 10 * 7 cm(longest * shortest, short scab/superficial/2 * 2 cm/ boot shape) wide/Skin alopecia rashes at groin 504 hr No obvious clinical Induration shrink/ alopecia observation 9 * 3 cm/some induration near the right knee, 0.2~0.5 deep/Skin rashes at groin 528 hr rashes at the vehcle Induration shrink/ alopecia injection area 9 * 3 cm/some induration near the right knee, 0.2~0.5 deep/Skin rashes at groin and oxter 552 hr rashes began to Induration shrink/ alopecia scab 8 * 3 cm/some induration near the right knee, 0.2~0.5 deep/Skin rashes at groin and oxter/scabs 0.8 * 0.6 cm 576 hr rashes began to Induration shrink/ alopecia scab 7 * 3 cm/some induration near the right knee, 0.2~0.5 deep/Skin rashes at groin/ scabs 0.8 * 0.6 cm 600 hr rashes began to Induration shrink/ alopecia scab 7 * 3 cm/some induration near the right knee, 0.2~0.5 deep/Skin rashes at groin and oxter/scabs 0.8 * 0.6 cm 624 hr rashes began to Induration shrink/ alopecia scab 7 * 3 cm/some induration near the right knee, 0.2~0.5 deep/Skin rashes at groin and oxter/scabs 0.8 * 0.6 cm 648 hr rashes began to Induration shrink/ No obvious clinical scab 7 * 3 cm/Skin rashes at groin observation and oxter 672 hr rashes Induration shrink/ rashes 5.5 * 3 cm/Skin rashes at groin and oxter 696 hr rashes Induration shrink/ rashes shrink 5 * 3 cm/Skin rashes at groin and oxter 720 hr rashes Induration shrink/ No obvious clinical 4.5 * 3 cm/Skin rashes at groin observation 744 hr rashes Induration shrink/ No obvious clinical 4 * 3 cm/Skin rashes at groin observation and oxter 768 hr rashes Induration shrink/ No obvious clinical 4 * 3 cm/Skin rashes at groin observation and oxter 792 hr rashes Induration shrink/ No obvious clinical 4 * 3 cm/Skin rashes at groin observation and oxter 816 hr rashes Induration shrink/ No obvious clinical 4 * 3 cm/Skin rashes at groin observation and oxter 840 hr rashes Induration shrink/ Swelling/red/warmth 4 * 3 cm/Skin rashes at groin and oxter/BT: 38.5° C. 864 hr rashes Induration shrink/ Swelling/red/warmth 4 * 3 cm/Skin rashes at groin and oxter/BT: 38.7° C. 888 hr rashes Induration shrink/ Swelling/red/warmth 4 * 3 cm/Skin rashes at groin and oxter/BT: 38.0° C. 912 hr rashes Induration shrink/ Swelling/red/warmth 3 * 2 cm/Skin rashes at groin and oxter/BT: 38.6° C. 936 hr rashes Induration shrink/ red/warmth 3 * 2 cm/Skin rashes at groin and oxter/BT: 38.2° C. 960 hr rashes Induration shrink/ No obvious clinical 3 * 2 cm/Skin rashes at groin observation and oxter/BT: 38.5° C. 984 hr rashes Induration shrink/ No obvious clinical 2 * 2 cm/Skin rashes at groin observation and oxter/BT: 38.3° C. 1008 hr  rashes Induration shrink/ No obvious clinical 2 * 2 cm/Skin rashes at groin observation and oxter/BT: 38.6° C. 1032 hr  rashes Skin rashes at groin and oxter No obvious clinical observation 1056 hr  rashes Skin rashes at groin and No obvious clinical oxter/BT: 38.3° C. observation 1080 hr  rashes Skin rashes at groin and oxter No obvious clinical observation 1104 hr  rashes Skin rashes at groin and No obvious clinical oxter/BT: 38.6° C. observation 1128 hr  rashes subsiding Skin rashes at groin and oxter No obvious clinical observation 1152 hr  rashes subsiding Skin rashes at groin and oxter No obvious clinical observation 1176 hr  rashes subsiding Skin rashes at groin and oxter No obvious clinical observation 1200 hr  rashes subsiding Skin rashes at groin and No obvious clinical oxter/BT: 38.7° C. observation 1224 hr  rashes subsiding Skin rashes at groin and No obvious clinical oxter/BT: 38.8 observation 1248 hr  No obvious clinical Skin rashes at groin and No obvious clinical observation oxter/BT: 38.9 observation 1272 hr  No obvious clinical Skin rashes at groin and oxter No obvious clinical observation observation 1296 hr  No obvious clinical Skin rashes at groin and oxter No obvious clinical observation observation 1512 hr  No obvious clinical Skin rashes at groin and oxter No obvious clinical observation observation D1002 D1003 time point R-TA L-vehicle R-TA  2 hr Swelling No obvious Swelling clinical observation  8 hr Larger swelling No obvious Larger swelling clinical observation  24 hr Larger swelling and a scab on No obvious Larger swelling the edge of the swelling part clinical observation  48 hr Larger swelling and a scab on No obvious Larger swelling the edge of the swelling part clinical observation  72 hr Larger swelling and a scab on No obvious Larger swelling the edge of the swelling part clinical observation  96 hr Larger swelling and a scab on No obvious Larger swelling the edge of the swelling part clinical observation 120 hr Larger swelling with No obvious Larger swelling induration and a scab on the clinical with induration edge of the swelling part observation 144 hr Larger swelling with No obvious Larger swelling induration and a scab on the clinical with induration edge of the swelling part observation 178 hr induration/5 * 8 cm wide/0.5-0.8 cm No obvious induration/5 * 5 cm depth; 0.3 * 0.3 cm/red clinical wide/0.5-1 cm scab observation depth 192 hr 0.3 * 0.1 cm/red scab No obvious induration/5 * 5 cm induration/5 * 8 cm wide/0.5-0.8 cm clinical wide/0.5-1 cm depth; 0.3 * 0.3 cm/red observation depth scab; 216 hr 0.2 * 0.1 cm/red No obvious Induration/5 * 8 cm scab/shrinking; clinical wide/0.5-1 cm Induration/4 * 8 cm wide/0.5-0.8 cm observation depth depth; two discolorer shin became yellow scab/1 * 1 cm, 2 * 2 cm 240 hr 0.2 * 0.1 cm/red No obvious Induration/5 * 7 cm scab/shrinking; clinical wide Induration/4 * 8 cm wide/; two observation discolorer shin became yellow scab/1 * 1 cm, 2 * 2 cm 264 hr 0.1 * 0.1 cm/red No obvious Induration/9 * 3.5 cm scab/shrinking; clinical wide/ Induration/7 * 7 cm wide/; two observation yellow scab/1 * 1 cm, 2 * 2 cm/superficial 288 hr Induration/7 * 8 cm/; two no significant Induration/9 * 5.5 cm yellow scab/1 * 1 cm, abnormal wide/ 2 * 2 cm/superficial 312 hr Induration/slight red/10 * 6 cm No obvious Induration/9 * 5 cm wide/0.5-0.8 cm depth; 3 clinical wide/0.5-1.5 cm yellow scabs/moderate; observation depth Skin discolored/red/3 * 2 cm/with some little red spot; 336 hr Induration/10 * 6 cm wide; 5 No obvious Induration/9 * 5 cm yellow clinical wide scabs(2 * 1 cm, 2 * 2 cm, 1 * 1 cm, observation 3 * 2 cm, 2 * 0.5 cm) on the right and one scab 2 * 2 on the left/moderate ulcer; 360 hr Induration/10 * 6 cm wide; 5 No obvious Induration/9 * 5 cm yellow clinical wide scabs(2 * 1 cm, 2 * 2 cm, 1 * 1 cm, observation 3 * 2 cm, 2 * 0.5 cm) on the right and one scab 2 * 2 on the left/alopecia 384 hr Induration, the edge of the No obvious Induration/9 * 5 cm induration became clinical wide soft/10 * 7 cm wide; 4 yellow observation scabs(1 * 1 cm, 2 * 2 cm, 3 * 2 cm, 2 * 0.5 cm) on the right and one scab 2 * 2 on the left/alopecia 408 hr Induration, the edge of the No obvious Induration/11 * 6 cm induration became clinical wide soft/10 * 7 cm wide; 4 yellow observation scabs(1 * 1 cm, 2 * 2 cm, 3 * 2 cm, 2 * 0.5 cm) on the right and one scab 2 * 2 on the left/alopecia 432 hr Induration, the edge of the No obvious Induration/10 * 5 cm induration became clinical wide soft/10 * 6 cm wide; 3 yellow observation scabs(1 * 1 cm, 3 * 2 cm, 2 * 0.5 cm) on the right and one scab 2 * 2 on the left/alopecia 456 hr Induration, the edge of the No obvious Induration/9 * 6 cm induration became clinical wide(oval) soft/11 * 7 cm wide(oval); 3 observation yellow scabs(1 * 1 cm, 3 * 2 cm, 2 * 0.5 cm) on the right and one scab 2 * 2 on the left/alopecia 480 hr Induration, the edge of the No obvious Induration/9 * 6 cm induration became clinical wide(oval) soft/11 * 7 cm wide(oval); 3 observation yellow scabs(1 * 1 cm, 3 * 2 and 2 * 0.5 cm) on the right and one scab 2 * 2 on the left/two red bumps/alopecia 504 hr Induration shrink, the edge of No obvious Induration the induration became clinical shrink/7 * 3 cm soft/9 * 6 cm wide; 2 yellow observation wide scabs(1 * 1 cm and 1.5 * 0.8 cm) on the right/two red bumps/alopecia 528 hr Induration shrink, the edge of No obvious Induration the induration became clinical shrink/7 * 3 cm soft/8 * 6 cm wide; 1 yellow observation wide scab(0.8 * 0.7 cm) on the right/two red bumps/alopecia 552 hr Induration shrink, the edge of No obvious Induration the induration became clinical shrink/7 * 3 cm soft/8 * 5 cm wide; 1 yellow observation wide scab(0.8 * 0.7 cm) on the right/ two red bumps/alopecia 576 hr Induration shrink, the edge of No obvious Induration the induration became clinical shrink/6 * 3 cm soft/7 * 5 cm wide; /two red observation wide bumps/alopecia 600 hr Induration shrink, the edge of No obvious Induration the induration became clinical shrink/6 * 3 cm soft/7 * 5 cm wide; /two red observation wide bumps/alopecia 624 hr Induration shrink, the edge of No obvious Induration the induration became clinical shrink/6 * 3 cm soft/7 * 5 cm wide; /two red observation wide bumps/alopecia 648 hr Induration shrink, the edge of No obvious Induration shrink the induration became clinical soft/7 * 5 cm wide; observation 672 hr Induration shrink, the edge of No obvious Swelling/6.5 * 3 cm the induration became clinical soft/7 * 4 cm wide; observation 696 hr Induration shrink, the edge of No obvious Swelling/5.5 * 3 cm the induration became clinical soft/6.5 * 4 cm wide; observation 720 hr Induration shrink, the edge of No obvious Swelling/5 * 3 cm the induration became clinical soft/5 * 4 cm wide; observation 744 hr Induration shrink, the edge of No obvious Swelling/5 * 3 cm/ the induration became clinical induration at the soft/5 * 4 cm wide; observation middle of swelling 768 hr Induration shrink, the edge of No obvious Swelling/5 * 3 cm/ the induration became clinical induration at the soft/5 * 4 cm wide; some observation middle of rushes were found at the swelling stomach 792 hr Induration shrink, the edge of No obvious Swelling/5 * 3 cm/ the induration became clinical induration at the soft/5 * 4 cm wide; some observation middle of rushes were found at the swelling stomach 816 hr Induration shrink, the edge of No obvious Swelling/4 * 3 cm/ the induration became clinical induration at the soft/5 * 3 cm wide; some observation middle of rushes were found at the swelling stomach 840 hr swelling for whole No obvious Swelling/4 * 3 cm/ leg/red/warmth/BT: 38.5° C. clinical rushes at observation groin/BT: 38.4° C. 864 hr swelling 5 * 3 cm at upper leg No obvious Swelling/4 * 3 cm/ and swelling for the whole clinical rushes at calf/red/warmth/BT: 38.9° C. observation groin/BT: 38.8° C. 888 hr swelling 5 * 3 cm at upper leg No obvious Swelling/4 * 3 cm/ and swelling for the whole clinical rushes at calf/red/warmth/BT: 38.5° C. observation groin/BT: 38.6° C. 912 hr swelling 5 * 3 cm at upper leg No obvious Swelling/4 * 3 cm/ and swelling for the whole clinical rushes at calf/red/warmth/BT: 38.9° C. observation groin/BT: 38.6° C. 936 hr swelling 3 * 2 cm at upper leg No obvious Swelling/3 * 2 cm/ and swelling for the whole clinical rushes at calf/red/warmth/BT: 38.9° C. observation groin/BT: 38.6° C. 960 hr swelling 3 * 2 cm at upper leg/ No obvious Swelling/3 * 2 cm/ BT: 39.0° C. clinical rushes at observation groin/BT: 38.8° C. 984 hr swelling 3 * 2 cm at upper leg/ No obvious Swelling/3 * 2 cm/ BT: 39.2° C. clinical rushes at observation groin/BT: 38.9° C. 1008 hr  swelling 3 * 2 cm at upper leg/ No obvious Swelling/3 * 2 cm/ BT: 38.9° C. clinical rushes at observation groin/BT: 38.6° C. 1032 hr  swelling 3 * 2 cm at upper leg No obvious Swelling/2 * 2 cm/ clinical rushes at groin observation 1056 hr  swelling 3 * 2 cm at upper leg/ No obvious Swelling/2 * 2 cm/ BT: 38.9° C. clinical rushes at observation groin/BT: 38.6° C. 1080 hr  swelling 3 * 2 cm at upper leg/ No obvious Swelling/2 * 2 cm/ BT: 38.8° C. clinical rushes at observation groin/BT: 38.9° C. 1104 hr  swelling 2 * 2 cm at upper leg/ No obvious swelling BT: 38.9° C. clinical shrinking/rashes observation subsiding/BT: 39.1° C. 1128 hr  swelling 2 * 2 cm at upper leg/ No obvious swelling BT: 38.8° C. clinical shrinking/rashes observation subsiding 1152 hr  swelling 2 * 2 cm at upper leg No obvious swelling clinical shrinking/rashes observation subsiding 1176 hr  swelling shrinking No obvious swelling clinical shrinking/rashes observation subsiding 1200 hr  swelling shrinking/BT: 38.8 No obvious swelling clinical shrinking/rashes observation subsiding/BT: 38.9° C. 1224 hr  swelling shrinking/BT: 39.0 No obvious swelling clinical shrinking/rashes observation subsiding/BT: 39.2 1248 hr  swelling shrinking/BT38.9 No obvious swelling clinical shrinking/rashes observation subsiding/BT: 38.8 1272 hr  No obvious clinical No obvious No obvious observation clinical clinical observation observation 1296 hr  No obvious clinical No obvious No obvious observation clinical clinical observation observation 1512 hr  No obvious clinical No obvious No obvious observation clinical clinical observation observation

Clinical Observations for Dogs Treated with Compound 24 at 48 mg/kg (DeepIM) are Provided in Table 75

D1501 D1502 D1502 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA  8 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation  24 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation/BT: observation observation observation observation 38.8  48 hr No obvious red spot near No obvious No obvious No obvious No obvious clinical the saphenous clinical clinical clinical clinical observation vein of hind observation observation observation observation limb/BT: 38.6  72 hr No obvious red spot near No obvious No obvious No obvious No obvious clinical the saphenous clinical clinical clinical clinical observation vein of hind observation observation observation observation limb/BT: 38.8  96 hr No obvious red spot near No obvious Swelling at the No obvious No obvious clinical the saphenous clinical whole leg, clinical clinical observation vein of hind observation induration observation observation limb/BT: 39.2 11 * 11 cm at the groin/red/BT: 38.3 120 hr No obvious red spot near No obvious Swelling at the No obvious No obvious clinical the saphenous clinical whole leg, clinical clinical observation vein of hind observation induration observation observation limb 11 * 11 cm at the subsiding/BT: groin/red/BT: 38.7 39.2 144 hr Red spot at Swelling No obvious Swelling at the No obvious swelling at the groin 8 * 5 cm at inner clinical whole leg, clinical inner upper leg upper leg with observation induration observation 8 * 8 cm/BT: 39.5 redness/swelling 11 * 11 cm at the at calf/red groin/red/BT: 38.3 spot near the saphenous vein of hind limb subsiding/BT: 39.6 168 hr Red spot at Swelling No obvious Swelling at the Red spot at swelling at the groin 8 * 5 cm at inner clinical whole leg, the groin inner upper leg upper leg with observation induration 8 * 8 cm/BT: 39.3 redness/swelling 11 * 11 cm at the at calf/red groin/red/BT: 38.4 spot near the saphenous vein of hind limb subsiding/BT: 39.6 192 hr Red spot at Swelling No obvious Swelling at the Red spot at swelling at the groin 8 * 5 cm at inner clinical whole leg, the groin inner upper leg upper leg with observation induration 9 * 9 cm 8 * 8 cm/BT: 38.0 redness/swelling at the at groin/red/BT: 38.5 calf/BT: 39.0 216 hr Red spot at swelling No obvious Swelling at the Red spot at swelling at the groin 8 * 5 cm/BT: 39.0 clinical whole leg, the groin inner upper leg observation induration 9 * 9 cm 8 * 8 cm/BT: 38.8 at the groin/red/BT: 38.4 240 hr Red spot at swelling No obvious Swelling 9 * 9 cm Red spot at swelling at the groin 8 * 5 cm/BT: 39.0 clinical at the back and the groin inner upper leg observation inner upper 8 * 8 cm/BT: 39.2 leg/red/BT: 38.9 264 hr Red spot at swelling No obvious Swelling 9 * 9 cm Red spot at swelling at the groin 6 * 5 cm/BT: 38.9 clinical at the back and the groin inner upper leg observation inner upper 8 * 8 cm/BT: 38.9 leg/red/BT: 38.3 288 hr Red spot at swelling No obvious Swelling 9 * 9 cm Red spot at swelling at the groin 6 * 5 cm/BT: 38.9 clinical at the back and the groin inner upper leg observation inner upper subsiding 8 * 8 cm/BT: 38.8 leg/red/BT: 38.5 day 13 Red spot at swelling No obvious Swelling 9 * 9 cm No obvious swelling at the groin 6 * 5 cm clinical at the back and clinical inner upper leg observation inner upper leg observation 8 * 8 cm day 14 Red spot at swelling No obvious Swelling 9 * 8 cm No obvious swelling at the groin 3 * 4 cm clinical at the back and clinical inner upper leg observation inner upper leg observation 8 * 7 cm day 15 Red spot at swelling No obvious Swelling 8 * 8 cm No obvious swelling at the groin 3 * 4 cm clinical at the back and clinical inner upper leg observation inner upper leg observation 8 * 7 cm day 16 Red spot at No obvious No obvious Swelling 6 * 6 cm No obvious swelling at the groin clinical clinical at the back and clinical inner upper leg observation observation inner upper leg observation 6 * 6 cm day 17 Red spot at No obvious No obvious Swelling 6 * 5 cm No obvious swelling at the groin clinical clinical at the back and clinical inner upper leg observation observation inner upper leg observation 4 * 5 cm day 18 Red spot at No obvious No obvious Swelling 6 * 5 cm No obvious swelling at the groin clinical clinical at the back and clinical inner upper leg observation observation inner upper leg observation 4 * 5 cm day 19 Red spot at No obvious No obvious Swelling 5 * 5 cm No obvious swelling at the groin clinical clinical at the back and clinical inner upper leg observation observation inner upper leg observation 4 * 5 cm day 20 Red spot at No obvious No obvious Swelling 5 * 5 cm No obvious swelling at the groin clinical clinical at the back and clinical inner upper leg observation observation inner upper leg observation 4 * 5 cm day 21 Red spot at No obvious No obvious Swelling 4 * 4 cm No obvious swelling at the groin clinical clinical at the back and clinical inner upper leg observation observation inner upper leg observation 3 * 3 cm day 22 Red spot at No obvious No obvious Swelling 4 * 4 cm No obvious swelling at the groin clinical clinical at the back and clinical inner upper leg observation observation inner upper leg observation 2 * 2 cm day 23 Red spot at No obvious No obvious Swelling 4 * 4 cm No obvious No obvious the groin clinical clinical at the back and clinical clinical observation observation inner upper leg observation observation day 24 Red spot at No obvious No obvious Swelling 4 * 3 cm No obvious No obvious the groin clinical clinical at the back and clinical clinical observation observation inner upper leg observation observation day 25 Red spot at No obvious No obvious Swelling 4 * 3 cm No obvious No obvious the groin clinical clinical at the back and clinical clinical observation observation inner upper leg observation observation day 28 Red spot at No obvious No obvious Swelling 3 * 3 cm No obvious No obvious the groin clinical clinical at the back and clinical clinical observation observation inner upper leg observation observation day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation day 42 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation day 49 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation day 56 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation day 63 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 45 at 30 mg/kg are Provided in Table 76

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA Day 0 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 1 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 4 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 5 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 6 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 14 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 28 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 42 No obvious No obvious No obvious No obvious several rashes several rashes clinical clinical clinical clinical on the on the outside observation observation observation observation outside and and inside of inside of left right thigh leg/several rashes on the left crus Day 43 No obvious No obvious No obvious No obvious several rashes several rashes clinical clinical clinical clinical on the on the outside observation observation observation observation outside and and inside of inside of left right thigh leg/several rashes on the left crus Day 46 No obvious No obvious No obvious No obvious several rashes several rashes clinical clinical clinical clinical on the on the outside observation observation observation observation outside and and inside of inside of left right thigh/fresh leg/several rashes on the rashes on the upper thigh left crus/fresh rashes on the upper thigh Day 48 No obvious No obvious No obvious No obvious several rashes several rashes clinical clinical clinical clinical on the outside on the outside observation observation observation observation of left of right leg/escharosis thigh/several of several rashes on the rashes on the thighs/escharosis left of several crus/several rashes on the rashes on the inside of the leg thighs Day 49 several rashes No obvious No obvious No obvious several rashes several rashes on the outside clinical clinical clinical on the outside on the outside and inside observation observation observation and inside and inside of of left of left right thigh leg/several leg/several rashes on rashes on the thighs the thighs Day 53 No obvious No obvious No obvious No obvious recovery for recovery for clinical clinical clinical clinical rashes on the rashes on the observation observation observation observation leg leg Day 55 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 56 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 45 at 48 mg/kg are Provided in Table 77

D1501 D1502 D1503 time point L-TA R-vehicle L-TA R-vehicle L-TA R-vehicle Day 0 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 1 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 14 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 28 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 42 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 49 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 56 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 63 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 70 No obvious No obvious No obvious No obvious Slight rashes on Slight rashes clinical clinical clinical clinical the outside of on the outside observation observation observation observation the hindlimb of the hindlimb. Day 73 No obvious No obvious No obvious No obvious Recovery for Recovery for clinical clinical clinical clinical rashes, skin rashes, skin observation observation observation observation exuviating exuviating Day 75 Slight rashes Slight rashes No obvious No obvious Recovery for Recovery for appeared on appeared on clinical clinical rashes, slight rashes, slight the outside of the outside of observation observation skin skin hindlimb. hindlimb. exuviating exuviating Day 77 Slight rashes Slight rashes New rashes New rashes New rashes New rashes appeared on appeared on appeared on appeared on appeared on appeared on the outside of the outside of the inside of the inside of both outside both outside hindlimb. hindlimb. the hindlimb. the hindlimb. and inside of and inside of the hindlimb. the hindlimb. Day 80 Slight rashes Slight rashes Slight rashes Slight rashes Recovery for Slight rashes appeared on appeared on on the inside on the inside the rashes on on the inside the outside of the outside of of the of the the outside and of the hindlimb. hindlimb. hindlimb, and hindlimb, and inside of the hindlimb, and new rashes new rashes hindlimb. recovery for appeared on appeared on the rashes on the outside. the outside. the inside Day 82 a few rash on No obvious a few rash a few rash rashes recovery for the outside of clinical on the on the disappeared on the rashes on left leg observation outside of outside of the inside and the outside leg/ leg leg outside of leg several rashes on the inside of leg Day 84 rashes No obvious escharosis of rashes rashes recovery for disappeared on clinical rashes on the disappeared disappeared on the rashes on the outside of observation outside of on the outside the inside and the outside leg/ left leg leg of leg outside of leg several rashes on the inside of leg Day 87 No obvious No obvious No obvious No obvious No obvious slight rashes clinical clinical clinical clinical clinical on the inside observation observation observation observation observation of leg Day 89 No obvious No obvious No obvious No obvious No obvious slight rashes clinical clinical clinical clinical clinical on the inside observation observation observation observation observation of leg Day 91 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 45 at 96 mg/kg are Provided in Table 78

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA Day 0 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 1 No obvious No obvious No obvious No obvious Liquid feces about 20 mL clinical clinical clinical clinical observation observation observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 4 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 5 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 6 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 14 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 28 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 35 No obvious No obvious Rashes on the Rashes and No obvious No obvious clinical clinical outside of the exuviate on clinical clinical observation observation hindlimb the outside observation observation of the hindlimb Day 38 Slight rashes Slight rashes Much rashes on Much rashes on No obvious No obvious on the inside on the outside the outside of the outside clinical clinical and outside of of the the hindlimb, of the observation observation the hindlimb hindlimb skin exuviating hindlimb, skin exuviating Day 40 Slight rashes Slight rashes Much rashes on Much rashes on No obvious No obvious on the inside on the outside the outside of the outside clinical clinical and outside of of the the hindlimb, of the observation observation the hindlimb hindlimb skin exuviating hindlimb, skin exuviating Day 42 Slight rashes Slight rashes Slight rashes on Much rashes on No obvious No obvious on the inside on the inside the outside of the outside clinical clinical and outside of and outside of the hindlimb, of the observation observation the hindlimb the hindlimb skin exuviating hindlimb, skin exuviating Day 45 Slight rashes Slight rashes Slight rashes on Much rashes on No obvious No obvious on the inside on the inside the outside of the outside clinical clinical and outside of and outside of the hindlimb, of the observation observation the hindlimb the hindlimb skin exuviating hindlimb, skin exuviating Day 47 Rashes Rashes Slight rashes on Much rashes on No obvious No obvious scabbing on scabbing on the outside of the outside clinical clinical the outside of the outside of the hindlimb, of the observation observation the hindlimb/ the hindlimb/ skin exuviating hindlimb, Slight rashes Slight rashes skin exuviating on the inside on the inside of the of the hindlimb hindlimb Day 49 rashes rashes Slight rashes on slight rashes on No obvious No obvious disappeared on disappeared on the outside of the outside of clinical clinical the inside and the inside and the hindlimb, the hindlimb, observation observation outside of the outside of the skin exuviating skin exuviating leg leg Day 52 No obvious rashes Slight rashes on slight rashes on No obvious No obvious clinical disappeared on the outside of the outside of clinical clinical observation the inside and the hindlimb, the hindlimb, observation observation outside of the skin exuviating skin exuviating leg Day 54 No obvious No obvious rashes rashes No obvious No obvious clinical clinical disappeared on disappeared on clinical clinical observation observation the outside of the outside of observation observation the hindlimb the hindlimb Day 56 No obvious No obvious No obvious rashes No obvious No obvious clinical clinical clinical disappeared on clinical clinical observation observation observation the outside of observation observation the hindlimb Day 59 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 7 at 24 mg/kg are Provided in Table 79

time D1001 D1002 point L-vehicle R-TA L-vehicle R-TA Day 0 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 1 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 2 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 3 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 4 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 5 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 6 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 7 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 14 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 21 No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation Day 28 Left hind leg The right hind leg a No obvious No obvious lateral small red few red rash, molting, clinical clinical rash, inside a small inside a small amount observation observation amount of red rash of red rash Day 30 Left hind leg Right leg lateral No obvious No obvious lateral small red have small red clinical clinical rash, inside a small rash, peeling observation observation amount of red rash Day 32 Left hind leg ; Right leg lateral No obvious No obvious lateral small red small red rash, clinical clinical rash, inside a lot of inside a lot of red observation observation red rash rash Day 35 Left hind leg ; Right leg lateral No obvious No obvious lateral small red small red rash, clinical clinical rash, inside a lot of inside a lot of red observation observation red rash rash Day 37 a small amount of exuviate on the No obvious No obvious exuviate on the outside of leg/a clinical clinical outside of leg/a lagre amount of rashes observation observation lagre amount of rashes on the inside of leg on the inside of leg Day 39 a small amount of exuviate on the No obvious No obvious exuviate on the outside of leg/a clinical clinical outside of leg/a lagre amount of rashes observation observation lagre amount of rashes on the inside of leg on the inside of leg Day 42 a lagre amount of a large amount of No obvious No obvious rashes on the inside rashes on the inside clinical clinical of leg of leg observation observation Day 44 a small amount of a small amount of No obvious No obvious rashes on the inside rashes on the inside clinical clinical of leg of leg observation observation Day 46 a small amount of a small amount of No obvious No obvious rashes on the inside rashes on the inside clinical clinical of leg of leg observation observation Day 49 a small amount of a small amount of No obvious No obvious rashes on the inside rashes on the inside clinical clinical of leg of leg, Red rash observation observation subsided

Clinical Observations for Dogs Treated with Compound 7 at 48 mg/kg are Provided in Table 80

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA  8 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation 24 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 14 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 28 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 42 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 49 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 56 No obvious No obvious No obvious No obvious several rashes several rashes clinical clinical clinical clinical on the on the observation observation observation observation left leg right leg Day 58 No obvious No obvious No obvious No obvious several rashes several rashes clinical clinical clinical clinical on the on the observation observation observation observation left leg right leg Day 61 No obvious No obvious No obvious No obvious several rashes several rashes clinical clinical clinical clinical on the on the observation observation observation observation left leg right leg Day 63 No obvious No obvious No obvious No obvious escharosis of several rashes clinical clinical clinical clinical several rashes on the observation observation observation observation on the outside outside of of left leg/ right leg several rashes on the inside of left leg Day 65 No obvious No obvious No obvious No obvious escharosis of recovery for clinical clinical clinical clinical rashes on the rashes on the observation observation observation observation outside of inside of left leg/rashes right leg subside on the inside of left leg Day 68 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 70 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 72 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 8 at 48 mg/kg are Provided in Table 81

D1501 D1502 D1503 L-vehicle L-vehicle L-vehicle (Sesameoil + (Cottonseed + (Sesameoil + 1% Benzyl 1% Benzyl 1% Benzyl time point Alcohol) R-TA Alcohol) R-TA Alcohol) R-TA  8 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation 24 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day l4 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 21 No obvious Swelling No obvious No obvious No obvious No obvious clinical 6 * 6.5 cm clinical clinical clinical clinical observation observation observation observation observation Day 28 No obvious Swelling No obvious No obvious No obvious No obvious clinical 5 * 3.5 cm clinical clinical clinical clinical observation observation observation observation observation Day 35 No obvious Swelling No obvious No obvious No obvious No obvious clinical 5 * 3 cm clinical clinical clinical clinical observation observation observation observation observation Day 37 Several rashes Several rashes No obvious No obvious No obvious No obvious on the inside on the outside clinical clinical clinical clinical and outside of the right observation observation observation observation of the left hindlimb. hindlimb. Swelling 5 * 3 cm. Day 40 Several rashes Several rashes No obvious No obvious No obvious No obvious on the inside on the inside clinical clinical clinical clinical and outside and outside observation observation observation observation of the left of the right hindlimb, more hindlimb, more on the inside. on the inside. Day 42 Several rashes Several rashes Several rashes Several rashes No obvious No obvious on the inside on the inside on the outside on the outside clinical clinical and outside and outside of the left of the right observation observation of the left of the right hindlimb hindlimb hindlimb, more hindlimb, more on the inside on the inside Day 44 Several rashes Several rashes Several rashes Several rashes No obvious One rash on on the inside on the inside on the outside on the outside clinical the outside of of the left and outside of the left of the right observation the right hindlimb of the right hindlimb hindlimb hindlimb hindlimb, more on the inside Day 47 Rashes on the Rashes on Rashes on the Rashes on the No obvious Rashes on inside of the left the outside outside of the outside of the clinical the outside of hindlimb were of the hindlimb were hindlimb were observation the hindlimb scabbing hindlimb disappearing disappearing were were disappearing disappearing, rashes on the inside were scabbing Day 49 Slight rashes Slight rashes Slight rashes Slight rashes No obvious No obvious appeared on the appeared on appeared on the appeared on the clinical clinical outside of the the outside outside of the outside of the observation observation hindlimb, rashes of the hindlimb hindlimb on the inside hindlimb, rashes were scabbing on the inside were scabbing Day 51 Slight rashes Slight rashes Slight rashes Slight rashes No obvious No obvious on the outside on the outside on the outside on the outside clinical clinical of the of the of the of the observation observation hindlimb, some hindlimb, hindlimb hindlimb, new rashes rashes on the rashes and appeared on the inside were exuviate inside scabbing appeared on the groin Day 54 Slight rashes Recovery Recovery Slight rashes No obvious No obvious on the outside for the for the on the outside clinical clinical of the rashes rashes of the observation observation hindlimb hindlimb, rashes and exuviate appeared on the groin, some new rashes appeared on the inside Day 56 Slight rashes New rashes Slight rashes Slight rashes No obvious No obvious and skin appeared on on the outside appeared on clinical clinical exuviating on the outside of the both inside observation observation the outside of of the hindlimb and outside the hindlimb. of the hindlimb, some hindlimb, new rashes rashes and skin appeared on the exuviating inside. on the groin. Day 58 Slight rashes Slight rashes Slight rashes Slight rashes No obvious No obvious and skin on the outside on the outside on both clinical clinical exuviating on of the of the inside and observation observation the outside of hindlimb hindlimb outside the of the hindlimb, slight hindlimb, rashes on the rashes and skin inside exuviating on the groin Day 61 Slight rashes Recovery for Slight rashes Slight rashes No obvious No obvious on the inside the rashes on on the outside on both clinical clinical of the the outside of the inside and observation observation hindlimb of the hindlimb, skin outside hindlimb exuviating of the hindlimb, skin exuviating on the outside, rashes and skin exuviating on the groin Day 63 Recovery for Recovery for Slight rashes on Slight rashes No obvious No obvious the rashes on the rashes on the outside on both clinical clinical the outside of the outside of the inside and observation observation the hindlimb/ of the hindlimb, skin outside of rashes on the hindlimb exuviating the inside of the hindlimb, skin hindlimb exuviating on the outside, rashes and skin exuviating on the groin Day 65 Recovery for Recovery for Slight rashes Slight rashes No obvious No obvious the rashes on the rashes on on the outside on both clinical clinical the outside of the outside of the inside and observation observation the hindlimb/ of the hindlimb, skin outside of the escharosis of hindlimb exuviating hindlimb, skin rashes on the exuviating inside of the on the hindlimb outside, rashes and skin exuviating on the groin Day 68 rashes scabbing No obvious Slight rashes on Slight rashes No obvious No obvious on the inside of clinical the outside on both clinical clinical the hindlimb observation of the inside and observation observation hindlimb, skin outside of the exuviating hindlimb, skin exuviating on the outside, rashes and skin exuviating on the groin Day 70 rashes scabbing No obvious No obvious rashes and No obvious No obvious on the inside of clinical clinical skin clinical clinical the hindlimb observation observation exuviating observation observation on the groin Day 72 rashes scabbing No obvious No obvious rashes and No obvious No obvious on the inside of clinical clinical skin clinical clinical the hindlimb observation observation exuviating observation observation on the inside of the hindlimb and groin Day 75 rashes No obvious No obvious slight rashes No obvious No obvious disappeared on clinical clinical and skin clinical clinical the inside of the observation observation exuviating observation observation hindlimb on the inside of the hindlimb and groin

Clinical Observations for Dogs Treated with Compound 1 at 48 mg/kg are Provided in Table 82

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA  8 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation 24 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 2 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 3 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 7 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 14 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 21 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 28 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 35 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 42 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 49 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 56 No obvious No obvious No obvious No obvious several rashes No obvious clinical clinical clinical clinical on the left leg clinical observation observation observation observation observation Day 58 No obvious No obvious No obvious No obvious several rashes No obvious clinical clinical clinical clinical on the outside clinical observation observation observation observation of left leg observation Day 61 No obvious No obvious No obvious No obvious several rashes No obvious clinical clinical clinical clinical and escharosis clinical observation observation observation observation on the outside observation of left leg Day 63 No obvious No obvious No obvious No obvious escharosis of No obvious clinical clinical clinical clinical rashes on the clinical observation observation observation observation outside of left observation leg Day 65 No obvious No obvious No obvious No obvious recovery for No obvious clinical clinical clinical clinical rashes on clinical observation observation observation observation the outside observation of left leg Day 68 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 70 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 72 No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation

Clinical Observations for Dogs Treated with Compound 3 at 48 mg/kg are Provided in Table 83

D1001 D1002 D1003 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA 8 hr No obvious No obvious No obvious Swelling No obvious Swelling 4 * 3 clinical clinical clinical 4 * 3.5 clinical observation observation observation observation Day 1 spotted swelling/4 * 4.5 No obvious Induration/7 * 5 spotted Induration/8 * 5.5 cm/ slight cm/BT: 39.2° C./ clinical cm/obvious slight obvious bulge/red red skin 2 * 2 cm observation bulge/BT: red skin skin/higher red spot at 39.1° C. temperature than left the edge of injection shaving area side/BT: 39.6° C. Day 2 swelling/5 * 6 Induration/8 * 11 * 1 Swelling/3 * 3 Induration/7 * 7 * 1.5 Swelling/3 * 3 Induration/8 * 5.5 * 1 cm/ cm/spotted cm/1 * 1 cm cm/obvious cm/spotted obvious bulge/red slight red red spot near bulge/BT: slight red skin skin/higher skin the injection 39.3° C. temperature than left site/3 * 3 injection red spot side/BT: 39.7° C. with yellow discharge at edge of shaving area/BT: 38.9° C. Day 3 swelling/5 * 6 Induration/8 * 11 * 1 Swelling/3 * 3 Induration/9 * 11 * 1.5 Swelling/3 * 3 Induration/7 * 11 * 1 cm/ cm/spotted cm/3 * 3 cm/spotted cm/obvious cm/spotted obvious bulge/red slight red red spot slight red skin bulge/BT: slight red skin skin/higher skin with yellow 39.3° C. temperature than left discharge at injection edge of side/BT: shaving 39.2° C. area/BT: 39.1° C. Day 4 swelling/5 * 6 Induration/10 * 11 * 1 Swelling/3 * 4 Induration/9 * 11 * 1.5 Swelling/3 * 3 Induration/8 * 12 * 1.5 cm/spotted cm/3 * 3 cm/spotted cm/obvious cm/spotted cm/obvious slight red red spot slight red skin bulge/BT: slight red skin bulge/red skin/ skin with yellow 38.5° C. higher temperature discharge at than left injection edge of side/another shaving induration 4 cm to area/BT: the injection 38.5° C. site/BT: 38.7° C. Day 5 swelling/5 * 6 Induration Swelling/2.5 * Induration/9 * 10 * 2 Swelling/2 * 3.5 Induration for the cm/spotted for the whole 3.5 cm/spotted cm/obvious cm/spotted whole upper slight red upper leg/ slight red skin bulge/slight slight red skin leg/slight swelling skin edema for warmth/BT: for the whole the whole 38.7° C. calf/another calf/ induration and tenderness, obverious bulge fluctuance, 4 cm to the injection warmth/1.5 * 2 site/3 * 3 cm red spot red spot behind the upper leg with yellow with slight discharge at swelling/BT: edge of 39.0° C. shaving area/BT: 38.6° C. Day 6 swelling/5 * 6 Induration Swelling/2 * 2 Induration/9 * 10 * 2 Swelling/2 * 3.5 Induration for the cm/spotted for the whole cm/spotted cm/obvious cm/spotted whole upper slight red upper leg slight red skin bulge/slight slight red skin leg/slight swelling skin/ with slight warmth/BT: for the whole swelling red/edema 38.8° C. calf/another for whole for the whole induration and calf calf/ obverious bulge tenderness, 4 cm to the injection fluctuance, site/3 * 3 cm red spot warmth/1.5 * 2 behind the upper leg red spot with slight with yellow swelling/BT: discharge at 39.2° C. edge of shaving area/BT: 38.4° C. Day 7 swelling/4 * 4 Induration Swelling/2 * 2 Induration/9 * 10 * 2 Swelling/2 * 3.5 Induration for the cm/spotted for the whole cm/spotted cm/obvious cm/spotted whole upper slight red upper leg slight red skin bulge/slight slight red skin leg/slight swelling skin/ with slight warmth/BT: for the whole swelling red/swelling 38.6° C. calf/another for whole for the whole induration and calf calf/red/ obverious bulge warmth/1 * 1 4 cm to the injection red spot with site/3 * 3 cm red spot yellow behind the upper leg discharge at with slight edge of swelling/BT: shaving 39.5° C. area/BT: 38.4° C. Day 8 swelling/4 * 4 Induration Swelling/2 * 2 Induration/9 * 10 * 2 Swelling/2 * 3.5 Induration for the cm/spotted for the whole cm/spotted cm/obvious cm/spotted whole upper slight red upper leg slight red skin bulge/slight slight red skin leg/slight swelling skin/ with slight warmth/BT: for the whole swelling red/swelling 38.7° C. calf/another for whole for the whole induration and calf calf/red/ obverious bulge warmth/1 * 1 4 cm to the injection red spot with site/3 * 3 cm red spot yellow behind the upper leg discharge at with slight edge of swelling/BT: shaving 39.4° C. area/BT: 38.7° C. Day 9 swelling/4 * 4 Induration Swelling Induration/9 * 10 * 2.5 Swelling/2 * 3.5 Induration/10 * 9 * 1.5 cm/spotted 11.5 * 9 cm at shrinking cm/obvious cm/spotted cm at the injection slight red the upper leg bulge/4 slight red skin site/slight swelling skin/ with slight scabs at 5 for the whole swelling red and o'clock 2 cm calf/another for whole warmth/slight near the induration and calf swelling for induration obverious bulge the whole and one fell 4 cm to the injection calf/BT: off/sight site/upper leg with 38.7° C. red/swelling slight at the whole swelling/BT: calf/BT: 38.8° C. 38.3° C. Day 10 swelling/4 * 4 Induration Swelling Induration/9 * 10 * 2.5 Swelling/2 * 3.5 Induration/10 * 9 * 1.5 cm/spotted 11 * 9 cm at shrinking cm/obvious cm/spotted cm at the injection slight red the upper leg bulge/4 slight red skin site/slight swelling skin/ with slight scabs at 5 for the whole swelling red and o'clock 2 cm calf/another for whole warmth/slight near the induration and calf swelling for induration obverious bulge the whole and two scabs 4 cm to the injection calf/BT: fell off/sight site/upper leg with 38.4° C. red/swelling slight at the whole swelling/BT: calf/BT: 38.3° C. 38.9° C. Day 11 swelling/4 * 4 Induration Swelling Induration/9 * 10 * Swelling Induration and cm/spotted 9 * 6 cm at the shrinking cm/3 warmth/10 * 9 * 1.5 cm slight red upper leg yellow scabs at the injection skin/ with slight at 5 o'clock site/another swelling red and 2 cm near the induration and for whole warmth/slight induration obverious bulge calf swelling for and two scabs 4 cm to the injection the whole fell off/sight site/upper leg with calf/BT: red/BT: slight 38.8° C. 38.9° C. swelling/BT: 39.4° C. Day 12 swelling/ Induration Swelling Induration/9 * 10 Swelling Induration and 4 * 4 cm 9 * 6 cm at the shrinking cm/3 warmth/10 * 9 * 1.5 cm upper leg yellow scabs at the injection with slight at 5 o'clock site/another red and 2 cm near the induration and warmth/BT: induration obverious bulge 38.0° C. and two scabs 4 cm to the injection fell off/sight site/3 * 3 cm red spot red/BT: behind the upper leg 38.5° C. with slight swelling/BT: 39.3° C. Day 13 swelling/ Induration Swelling Induration/9 * 10 Swelling Induration and 4 * 4 cm 9 * 5 cm at the shrinking cm/slight warmth/10 * 9 cm at upper leg red/warmth/BT: the injection with slight 38.8° C. site/another red and induration and warmth/BT: obverious bulge 38.4° C. 4 cm to the injection site/3 * 3 cm red spot behind the upper leg with slight swelling/BT: 39.9° C. Day 14 swelling/ Induration No obvious Induration/8 * 8 No obvious Induration and 4 * 4 cm 9 * 5 cm at the clinical cm/slight clinical warmth/8 * 9 cm at dosing site observation red/warmth/BT: observation the injection 38.3° C. site/another induration and obverious bulge 4 cm to the injection site/upper leg with slight swelling/BT: 39.6° C. Day 15 swelling/ Induration No obvious Induration/8 * 8 No obvious Induration and 4 * 4 cm shrinking clinical cm/slight clinical warmth/8 * 9 cm at 9 * 5 cm at the observation red/warmth/BT: observation the injection dosing site/BT: 37.8° C. site/another 37.8° C. induration and obverious bulge 4 cm to the injection site/upper leg with slight swelling/BT: 39.4° C. Day 16 swelling/ Induration No obvious Induration/8 * 8 No obvious Induration and 4 * 4 cm shrinking clinical cm/slight clinical warmth/8 * 9 cm at 9 * 5 cm at the observation red/warmth observation the injection dosing site site/slight swelling for the whole calf/another induration and obverious bulge 4 cm to the injection site/upper leg with slight swelling/BT: 39.6° C. Day 17 swelling/ Induration No obvious Induration/8 * 8 No obvious Induration and 4 * 4 cm shrinking clinical cm/BT: clinical warmth/8 * 9 cm at 9 * 5 cm at the observation 38.7° C. observation the injection site/ dosing site/ upper leg with BT: 38.3° C. slight swelling/BT: 39.5° C. Day 18 Swelling Induration No obvious Induration/8 * 8 No obvious Induration and shrinking shrinking clinical cm/BT: clinical warmth/8 * 9 cm at 9 * 5 cm at the observation 38.8° C. observation the injection site/ dosing site/ upper leg with BT: 38.4° C. slight swelling/BT: 39.5° C. Day 19 Swelling Induration No obvious Induration/8 * 8 No obvious Induration and shrinking shrinking clinical cm/BT: clinical warmth/8 * 9 cm at 9 * 5 cm at the observation 38.7° C. observation the injection site/ dosing site/ upper leg with BT: 38.3° C. slight swelling/BT: 39.4° C. Day 20 Swelling Induration No obvious Induration/8 * 8 No obvious Induration and shrinking shrinking clinical cm/BT: clinical warmth/8 * 9 cm at 9 * 5 cm at the observation 38.6° C. observation the injection site/ dosing site/ upper leg with BT: 37.9° C. slight swelling/BT: 39.6° C. Day 21 Swelling Induration No obvious Induration No obvious Induration and shrinking shrinking clinical shrinking/8 * 8 clinical warmth/8 * 9 cm at 9 * 5 cm at the observation cm/warmth/ observation the injection site/ dosing site/ BT: 38.5° C. upper leg with BT: 38.6° C. slight swelling/BT: 39.3° C. Day 22 Swelling Induration No obvious Induration No obvious Induration and shrinking shrinking clinical shrinking/8 * 8 clinical warmth/8 * 9 cm at 8 * 5 cm at the observation cm/warmth/ observation the injection site/ dosing site/ BT: 39.3° C. upper leg with BT: 38.8° C. slight swelling/BT: 39.9° C. Day 23 Swelling Induration No obvious Induration No obvious Induration and shrinking shrinking clinical shrinking/8 * 8 clinical warmth/8 * 9 cm at 8 * 5 cm at the observation cm/warmth/ observation the injection site/ dosing site/ BT: 38.8° C. upper leg with BT: 38.3° C. slight swelling/BT: 39.6° C. Day 24 No obvious Swelling No obvious Swelling No obvious Swelling and clinical 6 * 3 cm at the clinical shrinking/6 * 8 clinical warmth/6 * 6 cm at observation dosing site/ observation cm/warmth/ observation the injection site/ BT: 38.5° C. BT: 39.1° C. BT: 39.5° C. Day 25 No obvious Swelling No obvious Swelling No obvious Swelling and clinical 6 * 3 cm at the clinical shrinking/6 * 8 clinical warmth/6 * 6 cm at observation dosing site/ observation cm/warmth/ observation the injection site/ BT: 38.4° C. BT: 38.6° C. BT: 39.8° C. Day 26 No obvious Swelling No obvious Swelling No obvious Swelling 6 * 6 cm at clinical 5 * 3 cm at the clinical shrinking/ clinical the injection site/ observation dosing site/ observation 6 * 8 cm/ observation BT: 39.3° C. BT: 38.2° C. BT: 38.6° C. Day 27 No obvious Swelling No obvious Swelling No obvious Swelling 5 * 6 cm at clinical 5 * 3 cm at the clinical shrinking/6 * 6 clinical the injection site/ observation dosing site/ observation cm/BT: 38.6° C. observation BT: 38.9° C. BT: 38.4° C. Day 28 No obvious No obvious No obvious Swelling No obvious Swelling3 * 5 cm at clinical clinical clinical shrinking/3 * 5 clinical the injection site observation observation observation cm observation Day 29 No obvious No obvious No obvious Swelling No obvious Swelling3 * 3 cm at clinical clinical clinical shrinking/3 * 5 clinical the injection site observation observation observation cm observation Day 30 No obvious No obvious No obvious Swelling No obvious Swelling3 * 3 cm at clinical clinical clinical shrinking/3 * 3 clinical the injection site observation observation observation cm observation Day 31 No obvious No obvious No obvious Swelling No obvious No obvious clinical clinical clinical shrinking/3 * 3 clinical clinical observation observation observation cm observation observation Day 32 No obvious No obvious No obvious Swelling No obvious No obvious clinical clinical clinical shrinking/3 * 3 clinical clinical observation observation observation cm observation observation

Clinical Observations for Dogs Treated with Compound 4 at 48 mg/kg are Provided in Table 84

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA 8 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation Day 1 Slight Induration No obvious Swelling at the No obvious Induration swelling for the clinical upper leg 6 * 7 cm clinical for the whole leg/ observation observation whole leg warmth/lame Day 2 Slight Induration No obvious Swelling at the No obvious Induration swelling for the clinical upper leg 6 * 7 cm, clinical for the whole leg/ observation induration at the observation whole leg/warms warmth/lame inner upper leg 7 * 7 cm/warmth Day 3 Slight Induration No obvious Swelling at the No obvious Induration swelling for the clinical upper leg 6 * 7 cm, clinical for the whole leg/ observation induration at the observation whole leg/warms warmth/lame inner upper leg 7 * 7 cm/warmth Day 5 Slight Induration No obvious Swelling at the No obvious Induration swelling for the clinical upper leg 6 * 7 cm, clinical for the upper observation induration at the observation whole leg/warms leg/warmth inner upper leg 7 * 7 cm/warmth/scap 1 * 0.5 cm at the right of vulva Day 7 Slight Induration No obvious Swelling at the No obvious Induration swelling for the clinical upper leg 3 * 4 cm, clinical for the upper observation induration at the observation whole leg/warms leg/warmth inner upper leg 7 * 7 cm/warmth/scap 1 * 0.5 cm at the right of vulva Day 10 No obvious Induration No obvious Induration at the No obvious Induration clinical for the clinical upper leg/Scap at the clinical for the observation upper leg observation vulva fell off observation whole leg/warms Day 12 No obvious Induration No obvious Induration at the No obvious Induration clinical for the clinical upper leg clinical at the upper observation upper leg observation 7 * 4 cm/Scap at the observation leg 9 * 7 cm vulva fell off Day 17 No obvious Induration No obvious Induration No obvious Induration clinical for the clinical at the upper clinical at the upper observation upper leg observation leg 7 * 4 cm observation leg 9 * 6 cm Day 19 No obvious Induration No obvious Induration No obvious Induration clinical at the upper clinical at the upper clinical at the upper observation leg 8 * 5 cm observation leg 7 * 4 cm observation leg 7 * 4.5 cm Day 21 No obvious Induration No obvious Induration No obvious No obvious clinical at the upper clinical at the upper clinical clinical observation leg 8 * 4 cm observation leg 4 * 4 cm observation observation Day 24 No obvious Induration No obvious Induration No obvious No obvious clinical at the upper clinical at the upper clinical clinical observation leg 7.5 * 4 cm observation leg 3.5 * 4 cm observation observation Day 26 No obvious Induration No obvious No obvious No obvious No obvious clinical at the upper clinical clinical clinical clinical observation leg 6 * 4 cm observation observation observation observation Day 28 No obvious Induration No obvious No obvious No obvious No obvious clinical at the upper clinical clinical clinical clinical observation leg 6 * 4 cm observation observation observation observation Day 31 No obvious Induration No obvious No obvious No obvious No obvious clinical at the upper clinical clinical clinical clinical observation leg 6 * 4 cm observation observation observation observation Day 33 No obvious Induration No obvious No obvious No obvious No obvious clinical at the upper clinical clinical clinical clinical observation leg observation observation observation observation

Clinical Observations for Dogs Treated with Compound 5 at 48 mg/kg are Provided in Table 85

time D1001 D1002 point L-vehicle R-TA L-vehicle R-TA  72 hr No obvious Swelling Swelling Induration clinical 2 * 2.5 cm 2 * 3 cm 6.5 cm * 4.5 cm observation  96 hr No obvious Swelling Swelling Induration clinical 3 * 3 cm 4.5 * 2.5 cm 6 cm * 8 cm with observation slight red color 120 hr No obvious Swelling Swelling Induration clinical 3 * 3 cm 4.5 * 2.5 cm 6 cm * 8 cm with observation slight red color 144 hr No obvious Swelling Swelling Induration clinical 3.5 * 3 cm 4.5 * 2.5 cm 6 cm * 8 cm observation 168 hr No obvious Induration Swelling Induration clinical 4 * 3 cm 4.5 * 2.5 cm 6 cm * 8 cm observation 192 hr No obvious Induration Swelling Induration clinical 4 * 3 cm 4.5 * 2.5 cm 6 cm * 8 cm observation 216 hr No obvious Induration Swelling Induration clinical 4 * 3 cm 4.5 * 2.5 cm 6 cm * 8 cm observation 240 hr No obvious Swelling Swelling Induration clinical 2 * 3 cm/a 3.5 * 2 cm 7 cm * 8 cm observation swellen with pus at groin 264 hr No obvious No obvious Swelling Induration clinical clinical 2 * 2 cm 7 cm * 8 cm/warmth/ observation observation/ BT: 39.5° C. BT: 38.6° C. 288 hr No obvious No obvious Swelling Induration clinical clinical 2 * 2 cm 6 cm * 8 cm/ observation observation/ BT: 39.5° C. BT: 38.6° C. 312 hr No obvious No obvious Swelling Induration clinical clinical 2 * 2 cm shrinking/ observation observation/ 6 cm * 8 cm/ BT: 38.0° C. BT: 38.5° C. 336 hr No obvious No obvious Slight Induration clinical clinical swelling shrinking observation observation/ 2 * 2 cm 3 cm * 4 cm/ BT: 38.4° C. BT: 38.7° C. 360 hr No obvious No obvious No obvious Induration clinical clinical clinical shrinking observation observation/ observation 3 cm * 4 cm/ BT: 38.1° C. BT: 39.1° C. 384 hr No obvious No obvious No obvious Induration clinical clinical clinical shrinking observation observation/ observation 3 cm * 4 cm/ BT: 38.7° C. BT: 39.3° C. 408 hr No obvious No obvious No obvious Induration clinical clinical clinical shrinking observation observation observation 3 cm * 4 cm/ BT: 39.4° C. 432 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 38.9° C. BT: 38.0° C. 456 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation observation BT: 38.9° C. 480 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 39.2° C. BT: 38.0° C. 504 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation observation BT: 38.7° C. 528 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 37.9° C. BT38.2° C. 552 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 39.2° C. BT: 38.4° C. 576 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 39.0° C. BT: 38.4° C. 600 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 38.6° C. BT: 38.3° C. 624 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 38.9° C. BT: 38.6° C. 648 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 38.5° C. BT: 38.3° C. 672 hr No obvious No obvious No obvious Swelling clinical clinical clinical 3 cm * 3 cm/ observation observation/ observation BT: 39.3° C. BT: 38.3° C. 696 hr No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation 720 hr No obvious No obvious No obvious No obvious clinical clinical clinical clinical observation observation observation observation

Clinical Observations for Dogs Treated with Compound 35 at 48 mg/kg are Provided in Table 86

D1501 D1502 D1502 time point L-vehicle R-TA L-vehicle R-TA L-vehicle R-TA  8 hr No obvious No obvious No obvious No obvious No obvious No obvious clinical clinical clinical clinical clinical clinical observation observation observation observation observation observation  24 hr Swelling Red spots at groin Swelling No obvious Slight Slight swelling/red 4 * 4 cm 3 * 4 cm clinical swelling spot near the observation saphenous vein of hind limb  48 hr Swelling Red spots at groin Swelling No obvious Slight Slight swelling/red 4 * 4 cm 3 * 4 cm clinical swelling spot near the observation saphenous vein of hind limb  72 hr Swelling Swelling for the Swelling Swelling in the Swelling Swelling in the 4 * 4 cm whole leg; obvious 3 * 4 cm whole leg; obvious 5 * 5 cm whole leg; obvious swelling at upper leg swelling at swelling at upper with area of inner upper leg leg with area of 10 * 11 cm/Red spots with volume of 8 * 12 cm/red at the at groin/BT: 39.1° C. 15 * 13 * 3 cm and inner upper red/BT: 39.6° C. leg/BT: 39.3° C.  96 hr Swelling Swelling for the Swelling Swelling in the Swelling Swelling in the 4 * 4 cm whole leg; obvious 3 * 4 cm whole leg; obvious 5 * 5 cm whole leg; obvious swelling at upper leg swelling at swelling at upper with area of inner upper leg leg with area of 10 * 11 cm/Red spots with volume of 8 * 12 cm/ at groin/BT: 39.0° C. 15 * 13 * 3 cm and induration and red red/BT: 38.9° C. at the inner upper leg/BT: 39.3° C. 120 hr Swelling Swelling for the Swelling Swelling in the Swelling Swelling in the 4 * 4 cm whole leg; obvious 3 * 4 cm whole leg; obvious 5 * 5 cm whole leg; obvious swelling at upper leg swelling at swelling at upper with area of inner upper leg leg with area of 10 * 11 cm/Red spots with volume of 8 * 12 cm/ at groin/BT: 39.1° C. 15 * 13 * 3 cm and induration and red red/BT: 39.6° C. at the inner upper leg/red spot at calf/ BT: 39.4° C. 144 hr Swelling Obvious swelling at Swelling Induration in Swelling Swelling in the 4 * 4 cm upper leg with area 3 * 4 cm the whole leg; 5 * 5 cm whole leg; obvious of obvious swelling at upper 10 * 11 cm/induration swelling at leg with area of 5 * 4 cm at the back inner upper leg 8 * 12 cm/ of upper leg with with volume of induration and red redness/induration 15 * 13 * 3 cm and at the inner upper 9 * 7 cm at inner side redness/BT: 38.9° C. leg/red spot at calf/ of upper leg with BT: 39.0° C. redness/Red spots at groin/BT: 38.7° C. 168 hr Swelling Obvious swelling at Swelling Induration in Swelling Swelling in the 4 * 4 cm upper leg with area 3 * 4 cm the whole leg; 5 * 5 cm whole leg; obvious of obvious swelling at upper 10 * 11 cm/induration swelling at leg with area of 5 * 4 cm at the back inner upper leg 8 * 12 cm/ of upper leg with with volume of induration at the redness/induration 15 * 13 * 3 cm and back of upper leg 9 * 7 cm at inner side redness/BT: 39.9° C. with of upper leg with redness/induration redness/Red spots at 8 * 6 cm at the inner groin/BT: 38.9° C. upper leg/red spot at calf/BT: 39.3° C. 192 hr Swelling Obvious swelling at Swelling Induration in Swelling Obvious swelling 4 * 4 cm upper leg with area 3 * 4 cm the whole leg; 5 * 5 cm/red at upper leg with of obvious spots area of 8 * 12 cm/ 10 * 11 cm/induration induration at induration at the 5 * 4 cm at the back inner upper leg back of upper leg of upper leg with with 15 * 13 cm/ with redness/induration BT: 38.7° C. redness/induration 9 * 7 cm at inner side 8 * 6 cm at the inner of upper leg with upper leg/red spot redness/Red spots at at calf/BT: 39.2° C. groin/BT: 38.2° C. 216 hr Swelling Obvious swelling at Swelling Obvious Swelling induration at the 4 * 4 cm upper leg with area 3 * 4 cm induration at 4 * 4 cm/red back and inner of inner upper leg spots upper leg with 10 * 11 cm/induration with 13 * 9 cm/ 7 * 12 cm/red spot at 5 * 4 cm at the back BT: 39.2° C. calf/BT: 39.2° C. of upper leg with redness/induration 9 * 7 cm at inner side of upper leg with redness/Red spots at groin/BT: 38.6° C. 240 hr Swelling induration 5 * 4 cm at Swelling Obvious Swelling induration at the shrinking the back of upper 3 * 4 cm induration at 4 * 4 cm/red back and inner leg with inner upper leg spots upper leg with redness/induration with 13 * 9 cm/ 7 * 12 cm/red spot at 9 * 7 cm at inner side BT: 38.6° C. calf/BT: 38.9° C. of upper leg with redness/Red spots at groin/BT: 38.2° C. 264 hr Swelling induration 3 * 4 cm at Swelling Obvious Swelling induration at the shrinking the back of upper 3 * 3 cm induration at 2 * 2 cm/red back and inner leg with inner upper leg spots upper leg with redness/induration6 * 5 with 10 * 9 cm/ 6 * 6 cm/BT: 38.7° C. cm at inner side BT: 38.4° C. of upper leg with redness/Red spots at groin/BT: 38.0° C. 288 hr No obvious induration 3 * 4 cm at Swelling Obvious red spots induration at the clinical the back of upper 3 * 3 cm induration at back and inner observation leg with inner upper leg upper leg with redness/induration6 * 5 with 9 * 9 cm/ 6 * 6 cm/BT: 38.7° C. cm at inner side BT: 38.6° C. of upper leg with redness/Red spots at groin/BT: 38.4° C. day 13 No obvious induration 3 * 4 cm at No obvious Obvious No obvious induration at the clinical the back of upper clinical induration at clinical back and inner observation leg with observation inner upper leg observation upper leg with redness/Red spots at with 7 * 7 cm 6 * 6 cm groin day 14 No obvious induration 3 * 4 cm at No obvious Obvious No obvious induration at the clinical the back of upper clinical induration at clinical back and inner observation leg with observation inner upper leg observation upper leg with redness/Red spots at with 7 * 6 cm 6 * 6 cm groin day 15 No obvious induration 3 * 4 cm at No obvious Obvious No obvious induration at the clinical the back of upper clinical induration at clinical back and inner observation leg with observation inner upper leg observation upper leg with redness/Red spots at with 7 * 7 cm 6 * 6 cm groin day 16 No obvious induration 3 * 4 cm at No obvious Obvious No obvious induration at the clinical the back of upper clinical induration at clinical back and inner observation leg with observation inner upper leg observation upper leg with redness/Red spots at with 6 * 6 cm 6 * 6 cm groin day 17 No obvious induration 3 * 2 cm at No obvious Obvious No obvious induration at the clinical the back of upper clinical induration at clinical back and inner observation leg with observation inner upper leg observation upper leg redness/Red spots at with 6 * 6 cm with 4 * 6 cm groin day 18 No obvious induration 3 * 2 cm at No obvious Obvious No obvious induration at the clinical the back of upper clinical induration at clinical back and inner observation leg with observation inner upper leg observation upper leg redness/Red spots at with 5 * 5 cm with 4 * 3 cm groin day 19 No obvious redness/Red spots at No obvious Obvious No obvious induration at the clinical groin clinical induration at clinical back and inner observation observation inner upper leg observation upper leg with 4 * 5 cm with 2 * 3 cm day 20 No obvious redness/Red spots at No obvious Obvious No obvious Not obvious clinical groin clinical induration at clinical induration observation observation inner upper leg observation with 4 * 4 cm 

We claim:
 1. A compound, or pharmaceutically acceptable salt thereof, having a structure provided below:

wherein X is O or CH₂.
 2. A compound, or pharmaceutically acceptable salt thereof, wherein the compound is (((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl ((E)-octadec-9-en-1-yl) carbonate.
 3. A compound, or pharmaceutically acceptable salt thereof, wherein the compound is (((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl ((E)-octadec-9-en-1-yl) carbonate.
 4. A pharmaceutical composition comprising a compound of claim 1, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
 5. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable excipient is cottonseed oil.
 6. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable excipient is sesame oil.
 7. A pharmaceutical composition comprising a compound of claim 2, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
 8. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient is cottonseed oil.
 9. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient is sesame oil.
 10. A pharmaceutical composition comprising a compound of claim 3, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
 11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable excipient is cottonseed oil.
 12. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable excipient is sesame oil. 